Sjögren’s syndrome

treatment and treatment evaluation

© Jiska Marianne Meijer, 2010

All rights reserved.

No part of this publication may be reported or transmitted, in any form or by any means, without permis-

sion of the author.

Bookdesign: Saar de Vries, Studio Sgaar, Groningen

Printed by: Drukkerij van der Eems Heerenveen

ISBN: 978-90-367-4242-9

ISBN: 978-90-367-4241-2 (digitaal)

The research described in this thesis was financially supported by:

Roche Netherlands, NIH

The printing and distribution of this thesis was financially supported by:

Roche Nederland, Nationale Vereniging Sjögrenpatiënten, Nederlandse Vereniging voor Mondziekten en

Kaakchirurgie, Rijksuniversiteit Groningen, Groningen Graduate School of Medical Sciences, Reumafonds,

Henk van Dijk Tandtechniek, Tandtechnisch laboratorium Gerrit van Dijk, Fred Ribot tandtechniek, Synthes

(www.synthes.com), Martin Nederland, Van Velthuysen Liebrecht, Braun Medical B.V., Biomet 3i (www.

biomet3i.com), Henry Schein Dental (www.henryschein.nl), Dental Union, Smint powermints, Hoytema

Stichting

RIJKSUNIVERSITEIT GRONINGEN

Sjögren’s syndrometreatment and treatment evaluation

Proefschrift

ter verkrijging van het doctoraat in deMedische Wetenschappen

aan de Rijksuniversiteit Groningenop gezag van de

Rector Magniicus, dr. F. Zwarts,in het openbaar te verdedigen op

woensdag 12 mei 2010om 16.15 uur

door

Jiska Marianne Meijer

geboren op 6 maart 1979te Vlaardingen

Promotores: Prof. dr. A. Vissink

Prof. dr. C.G.M. Kallenberg

Copromotores: Dr. H. Bootsma

Dr. F.K.L. Spijkervet

Beoordelingscommissie: Prof. dr. J.C. Kluin-Nelemans

Prof. dr. I. van der Waal

Prof. dr. P.P. Tak

Paranimfen: Drs. W. Nesse

Drs. S.H. Visscher-Langeveld

Contents

chapter 1 Introduction

chapter 2

Health related quality of life, employment and disability in patients with Sjögren’s syndromeRheumatology. 2009 Sep;48(9):1077-82

chapter 3

The future of biologic agents in the treatment of Sjögren’s syndromeClin Rev Allergy Immunol. 2007 Jun;32(3): 292-7

Chapter 4 Tools for treatment evaluation

chapter 4a

Progression and treatment evaluation in diseases affecting salivary glandsIn: Wong DT. Salivary diagnostics. Ames (IA): Wiley-Blackwell; 2008. 214-25

chapter 4b

Salivary proteomic and genomic biomarkers for primary Sjögren’s syndromeArthritis Rheum. 2007 Nov; 56(11): 3588-600

Chapter 5 Treatment of primary Sjögren’s syndrome with rituximab

chapter 5a Treatment of primary Sjögren’s syndrome with rituximab: extended follow-up, safety and efficacy of retreatmentAnn Rheum Dis. 2009 Feb;68(2):284-5

chapter 5b Clinical and histological evidence of salivary gland restoration supports the efficacy of rituximab treatment in Sjögren’s syndromeArthritis Rheum. 2009 Oct 29;60(11):3251-6

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chapter 5c Effectiveness of rituximab treatment in primary Sjögren’s syndrome: a randomised, double-blind, placebo-controlled trialArthritis Rheum. 2010 Jan 13. (Epub ahead of print)

chapter 6 Sjögren’s syndrome and localized nodular cutaneous amyloidosis: Coincidence or a distinct clinical entity?Arthritis Rheum. 2008 Jul;58(7):1992-9

chapter 7 Summary and general discussion

chapter 8 Dutch summary

DankwoordCurriculum vitae

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Chapter 1

General introduction

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General introduction

The historical development of what currently is defined as Sjögren syndrome (SS) begins

with the description of Hadden in 1888 who noted an association between the presence

of a dry mouth and dry eyes in a 65-year old female patient who also suffered from loss

of taste and smell. When she was treated with a tincture of jaborandi (pilocarpine) three

times a day, her mouth became much more moist.(1) Also in 1888, Mickulicz described a

42-year old farmer with painless, extensive bilateral swelling of parotid and lacrimal glands.

The swelling disturbed his vision and interfered with eating. Mickulicz removed the greater

part of the swollen lacrymal glands. Unfortunately, a few months after surgery the patient

suddenly died, probably due to appendicitis. At that time, the diagnosis was not conclusive.

(2) However, the original woodcuts and colour plate of the drawing of a microscopical field

of the submandibular gland have been reviewed with our current knowledge and a diagnosis

of MALT lymphoma was made, a condition that rather frequently is observed in patients

with SS.(3)

In 1925 the French physician Gougerot related dry eyes and dry mouth to an exocrine

gland abnormality.(4) However, in 1933 Henrik Sjögren was the first to give a complete

description of the clinical and histological findings in patients with rheumatoid arthritis,

dry eyes and a dry mouth. In his thesis entitled ‘Zur Kentniss der Keratoconjuntivitis

sicca’ he presented clinical and pathological information of 19 cases of patients with such

complaints.(5) Sjögren stated that his major contribution has been the recognition of the

sicca syndrome as a systemic disease. At first there was a lot of criticism on his thesis and

only years later he received more credit for his work. His thesis was translated in English

by Hamilton in 1943.(6) The eponym Gougerot-Sjögren’s disease appeared in the literature

in the 1930-ies and was reduced to Sjögren’s disease a decade later due to the many cases

reported by Sjögren.

In 1965 Bloch described the same condition as a triad of keratoconjunctivitis sicca,

xerostomia and a connective tissue disease.(7) Based on this triad several sets of criteria

have been introduced in the eighties of the previous century,(8-11) but none of these

classification criteria were validated and universally accepted. In 1988 the European Study

Group on Classification Criteria for SS began a multicentre study in order to develop a

set of criteria.(12;13) This set of criteria received broader acceptance, although criticism

was raised as well. Therefore, a joint study of the European Study Group on Classification

Criteria for SS and a group of American experts was started. Presently, the revised

American-European classification criteria for SS, which were proposed in 2002, are the

most widely accepted and validated criteria (table 1).(14) These criteria combine subjective

symptoms of dry eyes and dry mouth with objective signs of keratoconjunctivitis sicca

and hyposalivation, and with serological and histopathological characteristics. It should be

mentioned that the revised American-European classification criteria for SS have not been

developed for clinical practice, but as a research tool for performing studies in patients

with SS. Nevertheless, they are now widely accepted as diagnostic tools for SS. One should

realize, however, that SS can be present in a patient who does not completely fulfil these

criteria.

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Table 1 Revised international classification criteria and revised rules for classification for SS (14).

I Ocular symptoms: a positive response to at least one of the following questions:

1. Have you had daily, persistent, troublesome dry eyes for more than 3 months?

2. Do you have a recurrent sensation of sand or gravel in the eyes?

3. Do you use tear substitutes more than 3 times a day?

II Oral symptoms: a positive response to at least one of the following questions:

1. Have you had a daily feeling of dry mouth for more than 3 months?

2. Have you had recurrently or persistently swollen salivary glands as an adult?

3. Do you frequently drink liquids to aid in swallowing dry food?

III Ocular signs-that is, objective evidence of ocular involvement defined as a positive result for a least one of the

following two tests:

1. Schirmer’s I test, performed without anaesthesia (≤5 mm in 5 minutes)

2. Rose Bengal score or other ocular dye score (≥4 according to Van Bijsterveld’s scoring system)

IV Histopathology: In minor salivary glands (obtained through normal-appearing mucosa) focal lymphocytic

sialoadenitis, evaluated by an expert histopathologist, with a focus score ≥1, defined as a number of lymphocytic

foci (which are adjacent to normal-appearing mucous acini and contain more than 50 lymphocytes) per 4 mm2

of glandular tissue

V Salivary gland involvement: objective evidence of salivary gland involvement defined by a positive result for at

least one of the following diagnostic tests:

1. Unstimulated whole salivary flow (≤ 1.5 ml in 15 minutes)

2. Sialectasia on parotid sialography

3. Abnormal salivary scintigraphy

VI Autoantobodies: presence in the serum of the following autoantibodies:

1. Antibodies to Ro(SSA) or La(SSB) antigens, or both

For primary SS

In patients without any potentially associated disease, primary SS may be defined as follows:

a) The presence of any 4 of the 6 items is indicative of primary SS, as long as either item IV (histopathol-

ogy) or VI (serology) is positive or

b) The presence of any 3 of the 4 objective criteria items (that is, items III, IV, V, VI)

For secondary SS

In patients with a potentially associated disease (for instance, another well defined connective tissue

disease), the presence of item I or item II plus any 2 from among items III, IV, and V may be considered

as indicative of secondary SS

Exclusion criteria

Past head and neck radiation treatment

Hepatitis C infection

Acquired immunodeficienty disease (AIDS)

Pre-existing lymphoma

Sarcoidosis

Graft versus host disease

Use of anticholinergic drugs (since a time shorter than 4-fold the half life of the drug)

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Although the first description of SS was given in 1888 and although SS is the second

autoimmune disease in prevalence (0.5-2%), only recently knowledge about SS has become

more generally recognized and over the last decades an increasing number of studies is

performed on SS. The first symptoms of SS usually develop gradually and are hard to

recognize without specific knowledge about SS. First symptom in almost all patients is

fatigue accompanied by one or more other symptoms such as oral and eye dryness,

arthralgia and extraglandular manifestations. Fortunately, SS is diagnosed more and more in

an early stage of the disease nowadays. Currently, more patients are within their working

age at the time of diagnosis (mean age 45.7±15.7 years).(15) The influence of having SS on

patients functioning and daily acitvity is still underestimated by both the general public and

physicians. Most patients with SS report a large impact of the disease on their quality of

life.(15) Moreover, related to the limitations patients experience in their daily life, there

is a growing request for treatment options, both from doctors and patients. Although, as

for other autoimmune diseases, the aetiopathogenesis of SS is still unknown, there are

indications that treatment with biological agents applied for other autoimmune diseases

might also be of benefit in the treatment of SS.(16) So far, B cell depletion showed the best

results amongst the biologicals tested.(17-20)

Before implementation of treatment of SS with biological agents can be realized, approval

should be obtained. Treatment with biological agents is expensive and positive impact on

socioeconomic status of SS patients should be clear before implementation. Biological

agents have to be investigated, first, in small open-label phase I trials to investigate safety

and efficacy and, thereafter, in double-blinded placebo controlled phase II trials and larger

phase III trials to confirm results found in the open-label trials. Also, research on the

aetiopathogenesis of SS is very important to gain more knowledge on the disease.

Although many trials have been performed during the last decades regarding treatment

of SS, including trials aimed at reducing disease activity and/or intervening with the

progression of the disease, up to now most agents that have been shown to be of some use

in the treatment of SS mainly exert a symptomatic effect. The assessment of the effect of

biologicals, aimed at reducing disease activity and to slow down progression of SS, is still at a

very early stage. Also, much remains unknown regarding the aetiopathogenesis of SS.

Therefore, the main objective of this thesis is the evaluation of existing and new

therapeutic strategies for intervention in SS. Furthermore, the impact of SS on quality of

life was assessed and a case report is described aiming to deepen the insight in the role of B

cells in the aetiology of SS.

Outline of this thesis

This thesis contains the results of various studies concerning (a) quality of life of SS

patients, (b) the applicability of tools to evaluate the efficacy of treatment in SS patients,

(c) the evaluation of intervention therapy with anti-CD20, a therapy that is focussed on

B cell depletion, and (d) a case series to gain more insight into the role of B cells in the

aetiopathogenesis of SS.

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The impact of SS on the quality of life and the socioeconomic status of SS patients is

described in chapter 2 This study was done to explore whether treatment is necessary for

SS patients and why research on this disease should be performed. Next, in chapter 3 a

specific overview of the trials performed up to 2006 with biological agents as treatment for

SS is given. The main conclusion from that overview is that anti-CD20 in particular seems to

be promising. In chapter 4a, a general overview of tools applicable for treatment evaluation

of diseases affecting salivary glands, in particular SS, is provided. On the basis of this

overview tools to be used in treatment evaluation (chapter 5) were selected. The possibility

of indentifying a genomic and proteonomic profile of SS patients as a new tool for evaluation

is described in chapter 4b. Based on the data published in chapters 2 and 3 and using a

selection of the tools provided in chapter 4, several trials with anti-CD20 (rituximab) as

intervention treatment for SS were designed. First, an analysis of the efficacy of retreatment

and long-term follow up after treatment (chapter 5a) is described. In chapter 5b a study is

presented evaluating the effects of rituximab on the histopathology of parotid gland biopsies

in patients with SS described in chapter 5a. Thereafter, a placebo controlled double blinded

randomized clinical trial of rituximab treatment in SS (chapter 5c) is described. A study

related to the direct scope of this thesis, is the description of a case series of 8 patients

in which the combination of nodular cutaneous amyloidosis and SS is present. (chapter 6)

The type of amyloid was probably AL amyloid in all 8 patients (immunoglobulin light chain-

associated amyloid). Therefore, the combination of cutaneous amyloid and SS appeared to

be a distinct disease entity reflecting a particular and benign part of the polymorphic spectre

of B cell dysfunction in lymphoproliferative diseases related to SS. Chapter 7 contains the

summary and general discussion and chapter 8 the Dutch summary.

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Reference List

(1) Hadden W.B. On “dry mouth” or suppresion of the salivary and buccal secretions. Transc Clin Soc

Lond 1888; 21:176.

(2) Mikulicz J.H. Uber eine eigenartige symmetrische Erkrankung der Tranen- und Mundspeicheldrusen.

Beitr Chir Fortschr Gewidmet Theodor Billroth, Stuttgart 1892;610-30.

(3) Ihrler S, Harrison JD. Mikulicz‘s disease and Mikulicz‘s syndrome: analysis of the original case

report of 1892 in the light of current knowledge identifies a MALT lymphoma. Oral Surg Oral Med

Oral Pathol Oral Radiol Endod 2005; 100(3):334-9.

(4) Gougerot A. Insuffisance progressive et atrophie des glandes salivaires et muqueuses, nasale,

laryngee, vulvaire. “Secheresse” de la bouche, des conjunctives, etc. Bull Soc Fr Derm Syph 1925;

32:376-9.

(5) Sjögren H.S. Zur Kentniss der Keratoconjuntivitis sicca (Keratitis filiformis bei Hypofunktion der

Tränendrüsen). Acta opthalmologica, Copenhagen; supplement II: 1-151. 1933.

(6) A new concept of kerato-conjunctivitis sicca. translated by J.B. Hamilton, in Australasian Medical,

Sidney. 1943.

(7) Bloch KJ, Buchanan WW, Wohl MJ, Bunim JJ. Sjögren‘s syndrome. A clinical, pathological, and

serological study of sixty-two cases. 1965. Medicine (Baltimore) 1992; 71(6):386-401.

(8) Fox RI, Robinson CA, Curd JG, Kozin F, Howell FV. Sjögren‘s syndrome. Proposed criteria for

classification. Arthritis Rheum 1986; 29(5):577-85.

(9) Homma M, Tojo T, Akizuki M, Yamagata H. Criteria for Sjögren‘s syndrome in Japan. Scand J

Rheumatol Suppl 1986; 61:26-7.

(10) Skopouli FN, Drosos AA, Papaioannou T, Moutsopoulos HM. Preliminary diagnostic criteria for

Sjögren‘s syndrome. Scand J Rheumatol Suppl 1986; 61:22-5.

(11) Manthorpe R, Oxholm P, Prause JU, Schiodt M. The Copenhagen criteria for Sjögren‘s syndrome.

Scand J Rheumatol Suppl 1986; 61:19-21.

(12) Vitali C, Bombardieri S, Moutsopoulos HM, Coll J, Gerli R, Hatron PY et al. Assessment of the

European classification criteria for Sjögren‘s syndrome in a series of clinically defined cases: results

of a prospective multicentre study. The European Study Group on Diagnostic Criteria for Sjögren‘s

Syndrome. Ann Rheum Dis 1996; 55(2):116-21.

(13) Vitali C, Bombardieri S, Moutsopoulos HM, Balestrieri G, Bencivelli W, Bernstein RM et al.

Preliminary criteria for the classification of Sjögren‘s syndrome. Results of a prospective concerted

action supported by the European Community. Arthritis Rheum 1993; 36(3):340-7.

(14) Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE et al. Classification

criteria for Sjögren‘s syndrome: a revised version of the European criteria proposed by the American-

European Consensus Group. Ann Rheum Dis 2002; 61(6):554-8.

(15) Meijer JM, Meiners PM, Huddleston Slater JJR, Spijkervet FKL, Kallenberg CG, Vissink A et al.

Health related quality of life, employment and disability in patients with Sjögren‘s syndrome.

Rheumatology (Oxford). 2009 sep;48(9):1077-82.

(16) Meijer JM, Pijpe J, Bootsma H, Vissink A, Kallenberg CG. The future of biologic agents in the

treatment of Sjögren‘s syndrome. Clin Rev Allergy Immunol 2007; 32(3):292-7.

(17) Dass S, Bowman SJ, Vital EM, Ikeda K, Pease CT, Hamburger J et al. Reduction of fatigue in Sjögren‘s

syndrome with rituximab: results of a randomised, double-blind, placebo controlled pilot study.

Ann Rheum Dis 2008; 67(11):1541-4.

(18) Devauchelle-Pensec V, Pennec Y, Morvan J, Pers JO, Daridon C, Jousse-Joulin S et al. Improvement

of Sjögren’s syndrome after two infusions of rituximab (anti-CD20). Arthritis Rheum 2007;

57(2):310-7.

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(19) Meijer JM, Pijpe J, van Imhoff GW, Vissink A, Spijkervet FK, Mansour K et al. Treatment of primary

Sjögren’s syndrome with rituximab: extended follow-up, safety and efficacy of retreatment. Ann

Rheum Dis 2009 Feb;68(2):284-5.

(20) Pijpe J, van Imhoff GW, Spijkervet FKL, Roodenburg JLN, Wolbink GJ, Mansour K et al. Rituximab

treatment in patients with primary Sjögren’s syndrome: An open-label phase II study. Arthritis

Rheum 2005; 52(9):2740-50.

Jiska M Meijer*1, Petra M Meiners*1, James JR Huddleston

Slater1,2, Fred KL Spijkervet1, Cees GM Kallenberg3, Arjan

Vissink1, Hendrika Bootsma3

* These authors contributed equally to this paper

Rheumatology. 2009 Sep;48(9):1077-82

Chapter 2

Health related quality of life,

employment and disability in patients

with Sjögren’s syndrome

1 Departments of Oral and Maxillofacial Surgery, 2 Oral Health Care and Clinical Epidemiology,

Academic Center for Oral Health, 3 Rheumatology and Clinical Immunology, University

Medical Center Groningen, University of Groningen, The Netherlands

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Abstract

Objective To compare health related quality of life (HR-QOL), employment and disability

between primary (pSS) and secondary (sSS) Sjögren’s syndrome (SS) patients and the

general Dutch population.

Methods HR-QOL, employment and disability were assessed in SS patients regularly

attending the University Medical Center Groningen (n=235). HR-QOL, employment and

disability were evaluated with the Short Form-36 questionnaire (SF-36) and an employment

and disability questionnaire. Results were compared with Dutch population data (matched

for sex and age). Demographical and clinical data associated with HR-QOL, employment

and disability were assessed.

Results Response rate was 83%. SS patients scored lower on HR-QOL than the general

Dutch population. sSS patients scored lower on physical functioning, bodily pain and ge-

neral health than pSS patients. Predictors for reduced HR-QOL were fatigue, tendomyalgia,

articular involvement, use of artificial saliva, use of antidepressants, comorbidity, male sex,

and eligibility for disability compensation (DC). Employment was lower and DC rates were

higher in SS patients compared with the Dutch population.

Conclusions SS has a large impact on HR-QOL, employment and disability.

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Introduction

Sjögren’s syndrome (SS) is a chronic, systemic, lymphoproliferative autoimmune disease

affecting the exocrine glands.(1) The salivary and lachrymal glands are most commonly

affected, resulting in dry mouth and dry eyes. Extraglandular involvement can occur in SS, and

includes, amongst others, pulmonary disease, renal disease and vasculitis. Moreover, almost

all patients suffer from fatigue. SS can be primary (pSS) or secondary (sSS), the latter being

associated with other autoimmune diseases such as rheumatoid arthritis (RA) or systemic

lupus erythematosus (SLE). The estimated prevalence of SS in the general population is

between 0.5-2%, which makes SS, after RA, the most common systemic autoimmune disease.

(2;3)

Rheumatologic conditions have a major impact on patients. Apart from the symptoms

mentioned above, patients may be restricted in their activities and their participation in

society, resulting in a reduced health related quality of life (HR-QOL) and an impaired

socioeconomic status. The latter may result in lower employment rates and more disability

as compared with the general population.(4)

SS is known to affect patients’ physical, psychological and social functioning (5), but

the impact of SS on HR-QOL, and especially on employment and disability, has not been

studied extensively. Studies available were either performed in small series of SS patients

(6;7) or mainly aimed at comparison with other rheumatic diseases (6-9), fatigue (9) and

psychological status (8), or at developing new tools for measuring fatigue and general

discomfort in pSS patients.(10) Comparison between pSS and sSS has occasionally been

described for HR-QOL (7;9), but not for employment and disability. The aim of this study

was, therefore, to evaluate HR-QOL, employment and disability in a large cohort of Dutch

SS patients, to relate outcomes to clinical and demographic data in this patient cohort, and

to compare these data with those available for the general Dutch population. In addition,

HR-QOL, employment and disability were compared between pSS and sSS patients, since

it was hypothesized that the disease burden of sSS might differ from that of pSS due to co-

existing autoimmune disease(s).

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Patients and methods

Patients

SS patients (185 pSS, 50 sSS) regularly attending the departments of Rheumatology, Clinical

Immunology and Oral and Maxillofacial Surgery of the University Medical Center Groningen

(UMCG), The Netherlands, were enrolled in this study. All patients were above the age of

18 years and fulfilled the European-American criteria for SS.(11) All patients participating

in this study were followed according to protocol, and, therefore, data on extraglandular

manifestations (EGM) were available for all patients. Ethical approval for this study was

obtained from the local Institutional Review Board.

Methods

Demographical and clinical data were obtained by chart review. EGM were defined in

accordance with previous studies.(12;13) Tendomyalgia, skin involvement other than

cutaneous vasculitis, oesophageal involvement, bladder involvement and thrombocytopenia

are commonly observed symptoms and signs, and, thus, were also considered as EGM.

Moreover, at every visit the rheumatologists systematically evaluated the presence of

EGMs.

Questionnaires were sent by regular mail to all patients. Six weeks after sending the

questionnaires, patients who had not responded were approached by phone once, to ask

for participation.

In the first questionnaire, patients were asked whether they suffered from arthralgia and/

or tendomyalgia, fatigue, dry mouth and dry eyes. In addition, it was asked which symptom

they considered to be their most severe complaint.

To evaluate HR-QOL, a validated Dutch translation of the Short Form 36 (SF-36) was

used.(12;14) The SF-36 is a questionnaire consisting of 36 items, with eight scales assessing

two dimensions, viz. physical and mental health functioning. Scales and summary scores vary

from 0 to 100, with 0 being the worst possible health status and 100 representing the best

possible health status.

The third questionnaire focused on level of education, employment and disability. In The

Netherlands, an individual who is judged to be impaired by at least 80% is entitled to full

disability compensation (DC). Individuals impaired by 15-80% are entitled to partial DC.

Age and sex matched data for the general Dutch population on the SF-36 were obtained

from Aaronson et al.(14) Data regarding employment and DC were obtained from the Dutch

Office of Statistics (Centraal Bureau voor Statistiek, CBS, Voorburg, The Netherlands).

Statistical analysis

The T-tests and χ2 tests were used for the comparison of demographical data, HR-QOL,

employment and receiving DC between responders and non-responders, between pSS and

sSS patients, and between SS patients and the general Dutch population. Alpha was set

at 5%. Correlation between disease duration and HR-QOL was evaluated with a Pearson

correlation test.

To create effect models, univariate analyses were performed for each predictor variable

on the outcomes (HR-QOL, employment and receiving DC). If variables were found to

be significant, P-values were used in the further development of the model. Predictors

with a P-value less than or equal to 0.2 were simultaneously entered into a multivariable

model, after which backward elimination of predictors was used to remove non-significant

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Table 1 Patients’ characteristics.

Characteristics All responding SS patients(n=195)

pSS(n=154)

sSS(n=41)

PpSS vs sSS

Age (years; mean±SD) 55.5±15.0 54.6±15.1 58.9±14.2 0.103

Age at diagnosis (years; mean±SD) 45.7±15.7 45.5±15.3 46.5±17.1 0.715

Female sex (n, %) 179 (91.8%) 143 (92.9%) 36 (87.8%) 0.197

Partner (n, %) 153 (78.5%) 121 (78.6%) 32 (78.0%) 0.769

Disease duration (years; mean±SD) 9.7±8.8 9.0±8.0 12.5±11.0 0.121

Immunological features

Focus score (mean±SD)

ANA (n, %)

Anti-Ro/SS-A (n, %)

Anti-La/SS-B (n, %)

IgG (g/L ; mean±SD)

IgA (g/L; mean±SD)

IgM (g/L; mean±SD)

RF (klU/L; mean±SD)

2.7±1.8

189 (96.9%)

155 (79.5%)

107 (54.9%)

18.6±7.2

2.8±1.3

1.4±1.0

106.2±190.2

2.7±2.0

151 (98.1%)

129 (83.8%)

90 (58.4%)

18.8±6.8

2.7±1.2

1.4±1.1

99.5±195.6

2.5±2.0

38 (92.7%)

26 (63.4%)

17 (41.5%)

17.7±8.3

3.2±1.5

1.3±0.8

131.2±168.7

0.716

0.109

0.014

0.077

0.405

0.023

0.629

0.343

Second auto immune disease (n, %)

none

SLE

RA

Other

154 (79.0%)

19 (9.7%)

16 (8.2%)

6 (3.1%)

154 (100%)

-

-

-

-

19 (46.3%)

16 (39.0%)

6 (14.6%)

-

-

-

-

Extraglandular manifestations (n, %)

Articular involvement*

Raynaud’s phenomenon

Tendomyalgia

Pulmonary involvement

Lymphoproliferative disease

Cutaneous vasculitis

Peripheral neuropathy

Skin involvement other than

cutaneous vasculitis*

Bladder involvement

Lymphadenopathy

Renal involvement

Autoimmune thyroiditis

Autoimmune hepatitis

Oesophageal involvement

Fever

Serositis

Myositis

CNS involvement

Thrombocytopenia

Acute pancreatitis

185 (94.9%)

110 (56.4%)

84 (43.1%)

80 (41.0%)

33 (16.9%)

30 (15.4%)

28 (14.4%)

26 (13.3%)

22 (11.3%)

22 (11.3%)

21 (10.8%)

19 (9.7%)

19 (9.7%)

12 (6.2%)

9 (4.6%)

8 (4.1%)

6 (3.1%)

5 (2.6%)

5 (2.6%)

2 (1.0%)

1 (0.5%)

144 (93.5%)

80 (51.9%)

67 (43.5%)

64 (41.6%)

25 (16.2%)

24 (15.6%)

22 (14.3%)

20 (13.0%)

13 (8.4%)

18 (11.7%)

19 (12.3%)

14 (9.1%)

16 (10.4%)

11 (7.1%)

7 (4.5%)

7 (4.5%)

5 (3.2%)

3 (1.9%)

5 (3.2%)

2 (1.3%)

1 (0.6%)

41 (100%)

30 (73.2%)

17 (41.5%)

16 (39.0%)

8 (19.5%)

6 (14.6%)

6 (14.6%)

6 (14.6%)

9 (22.0%)

4 (9.8%)

2 (4.9%)

5 (12.2%)

3 (7.3%)

1 (2.4%)

2 (4.9%)

1 (2.4%)

1 (2.4%)

2 (4.9%)

-

-

-

0.112

0.017

0.789

0.746

0.631

0.869

0.967

0.794

0.047

0.719

0.168

0.560

0.548

0.262

0.872

0.541

0.785

0.295

0.241

0.337

-

This table continues on the next page.

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Table 1 Patients’ characteristics, continued.

Characteristics All responding SS patients(n=195)

pSS(n=154)

sSS(n=41)

PpSS vs sSS

Comorbidity (n, %)** 75 (38.5%) 59 (38.3%) 16 (39.0%) 0.957

Osteoarthritis 15 (7.7%) 13 (8.4%) 2 (4.9%) -

Cardiovascular disease 13 (6.7%) 9 (5.8%) 4 (9.8%) -

Neurologic disease 10 (5.1%) 9 (5.8%) 1 (2.4%) -

Diabetes mellitus 8 (4.1%) 5 (3.2%) 3 (7.3%) -

Pulmonary disease 7 (3.6%) 5 (3.2%) 2 (4.9%) -

Gastro-intestinal disease 6 (3.1%) 5 (3.2%) 1 (2.4%) -

Eye disease 5 (2.6%) 4 (2.6%) 1 (2.4%) -

Malignancy 3 (1.5%) 3 (1.9%) 0 -

Urologic disease 3 (1.5%) 2 (1.0%) 1 (2.4%) -

Osteoporosis 2 (1.0%) 1 (0.6%) 1 (2.4%) -

Depression

Other

19 (9.7%)

10 (5.2%)

15 (9.7%)

6 (3.9%)

4 (9.8%)

4 (9.8%)

-

-

Therapy (n, %)

Artificial tears

Oral moisturising gel

Artificial saliva

Pilocarpine

NSAIDs

Antimalarial drugs

Oral corticosteroids

Rituximab

Other immunosuppressives

Antidepressants

151 (77.4%)

46 (23.6%)

20 (10.3%)

18 (9.2%)

47 (24.1%)

31 (15.9%)

26 (13.3%)

20 (10.3%)

17 (8.7%)

18 (9.2%)

119 (77.3%)

37 (24.0%)

16 (10.4%)

15 (9.7%)

31 (20.1%)

20 (13.0%)

20 (13.0%)

19 (12.3%)

9 (5.8%)

14 (9.1%)

32 (78.0%)

9 (22.0%)

4 (9.8%)

3 (7.3%)

16 (39.0%)

11 (26.8%)

6 (14.6%)

1 (2.4%)

8 (19.5%)

4 (9.8%)

0.711

0.840

0.942

0.663

0.012

0.031

0.783

0.036

0.006

0.769

n = number of patients; SLE = systemic lupus erythematosus; RA = rheumatoid arthritis; CNS = central

nervous system; NSAIDs = non-steroidal anti-inf lammatory drugs. *Extraglandular manifestation that

affect sSS patients significantly more frequently than pSS patients. **Comorbidity unrelated to SS.

predictors (P-value to remove >0.10). Subsequently, predictors not included in the

multivariable model were entered to determine whether they could now enter the model,

after which the procedure of backward elimination of predictors was repeated. Variables in

the final models were tested for possible interactions. All analyses were carried out using

SPSS for Windows version 16.0.

Results

Patient characteristics (table 1)

196 patients (180 females, 16 males; mean age at diagnosis: 45.7±15.7 years) responded

to the mail survey (83%). One patient returned the questionnaire incompletely and was

therefore excluded. The mean age (±SD) at the time of completing the questionnaire was

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55.5±15.0 years; the mean disease duration was 9.7±8.8 years. 154 patients (79%) were

classified as pSS and 41 patients (21%) as sSS. Demographic data did neither differ between

pSS and sSS patients nor between responders and non-responders.

The most frequently associated autoimmune disorders in sSS patients were SLE (46%)

and RA (39%). Seventy-five patients (39%) suffered from at least one comorbid condition.

Artificial tears were used by 77% and antidepressants by 9% of patients. Non-steroidal

anti-inflammatory drugs, antimalarial drugs and other immuno-suppressants were used more

frequently by sSS patients, whereas rituximab was more frequently prescribed in pSS patients.

EGM were present in 185 patients (95%). The main EGM were articular involvement,

Raynaud’s phenomenon and tendomyalgia. sSS patients suffered from articular- and skin

involvement more often than pSS patients. When restricting the EGM to the EGM defined

in accordance with previous studies (21;22), EGM occurred in 177 patients (91%; pSS 137;

sSS 40).

Current symptoms

Almost all patients suffered from dry mouth (n=183; 94%), dry eyes (n=183; 94%), and

fatigue (n=166; 85%). Fatigue was the most severe symptom in 78 patients (40%). Arthralgia

and/or tendomyalgia was present in 148 patients (76%). The prevalence of sicca symptoms,

fatigue and arthralgia and/or tendomyalgia was comparable between pSS and sSS patients.

Health related quality of life

When compared with the general Dutch population, HR-QOL was significantly decreased

in SS patients as demonstrated by reduced SF-36 scores on six out of the eight scales and

for the summary scores for physical and mental functioning (table 2).

sSS patients experienced a significantly lower HR-QOL than pSS patients on three of

the four physical scales (physical functioning, bodily pain and general health), however, no

differences were observed on the psychological scales. HR-QOL was comparable between

sSS patients with either RA or SLE as the associated autoimmune disorder. Disease duration

was not significantly correlated with any of the SF-36 scales. Highly educated patients

scored significantly better on physical functioning (p=0.042) and mental health (p=0.005)

compared with non-highly educated patients.

Multivariate regression analysis showed that fatigue, tendomyalgia, comorbidity, male

sex and receiving DC were associated with a reduced physical component summary score

(PCS) (table 3). Confounders were disease duration, use of NSAIDs and antidepressants

and employment. No significant effect modifiers (interaction terms) were found.

Multivariate regression analysis for the mental component summary score (MCS) de-

monstrated that fatigue, articular involvement, use of artificial saliva, use of antidepressants,

and comorbidity were associated with a reduced MCS, whereas dry mouth was associated

with a higher MCS (table 3). Receiving DC was a confounding factor for the determinants in

the primary model for the MCS. No effect modifiers were found.

Socioeconomic status

135 patients (69%) were of working age (18-65 years) (table 4). SS patients were significantly

less often employed (p<0.001), worked fewer hours (p=0.015) and were less frequently full

time employed (p<0.01), compared with the Dutch population. In detail, approximately half of

the SS patients (n=69) had paid employment. Only seven SS patients (10%) worked full-time

(at least 36 hours). On average, SS patients worked 21.7±11.6 hours per week. The mean sick

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Table 2 SF-36 scores for SS patients and the general Dutch population.

SF-36scales and summary scores

GDPMean (SD)n=195

RSS Mean (SD)n=195

PRSS vs GDP

pSSMean (SD)n=154

sSSMean (SD)n=41

PpSS vs sSS

PF

RP

BP

GH

VT

SF

RE

MH

PCS

MCS

74.8 (25.8)

70.3 (36.3)

68.7 (25.6)

65.7 (21.5)

63.8 (21.0)

81.3 (25.6)

79.7 (34.4)

73.3 (19.0)

73.0 (24.6)

74.5 (21.1)

59.2 (26.0)

41.0 (42.9)

64.7 (24.4)

40.3 (18.2)

45.2 (20.1)

63.1 (26.2)

70.0 (41.4)

70.3 (18.4)

51.7 (23.7)

63.3 (21.2)

0.000

0.000

0.136

0.000

0.000

0.000

0.005

0.055

0.000

0.000

62.0 (25.1)

44.0 (42.7)

68.0 (23.0)

41.9 (18.4)

46.0 (20.4)

64.5 (26.6)

71.5 (41.5)

70.6 (18.9)

53.3 (23.6)

64.0 (21.2)

48.9 (27.0)

29.1 (41.9)

52.1 (25.7)

34.2 (16.3)

42.0 (18.9)

57.9 (24.5)

63.9 (40.9)

69.0 (16.8)

44.7 (23.2)

60.5 (21.4)

0.004

0.058

0.000

0.018

0.266

0.152

0.324

0.627

0.055

0.385

n = number of patients; SF-36 = short form-36; GDP = general Dutch population; RSS = all responding SS

patients; pSS primary Sjögren syndrome; sSS = secondary Sjögren syndrome; PF = physical functioning;

RP = physical role functioning; BP = bodily pain; GH = general health; VT = vitality; SF = social function-

ing; RE = emotional role functioning; MH = mental health; PCS = physical component summary score;

MCS = mental component summary score.

leave was 15.6±39.0 days during the past year (range 0-192 days). Highly educated patients

were significantly more often employed than non-highly educated patients (p=0.001). No

differences were found between pSS and sSS patients regarding employment variables.

Sixty-three working age patients (47%) received DC, because they were considered to

be (partially) unfit for work (table 4). 28 of these patients (44%) were entitled to full DC.

Moreover, 41 of the 63 patients receiving DC (65%) mentioned pSS, sSS or the associated

rheumatic disease as the cause of receiving DC. No differences in DC were found between

pSS and sSS patients or between highly educated and non-highly educated patients. A

significantly higher percentage of SS patients received DC (47%) when compared with the

general Dutch population (2%).

Multivariate regression analysis for employment (table 5) showed that a high level of

education was associated with employment. Bladder involvement, use of oral moisturizing

gel, NSAIDs and oral corticosteroids, comorbidity and age at diagnosis were all negatively

associated with employment. Autoimmune thyroiditis, use of artificial tears and age were

confounding factors for these determinants. No interaction terms were found. Multivariate

regression analysis for receiving DC (table 5) demonstrated that the number of EGM, use

of artificial saliva and antimalarial drugs, comorbidity, high level of education, and male sex

were associated with receiving DC. Age at diagnosis was negatively associated with recei-

ving DC. Fatigue, skin involvement other than cutaneous vasculitis and use of pilocarpine

were confounding factors for the determinants in the primary model for receiving DC. No

interaction terms were found.

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Discussion

This study shows that SS has a large impact on HR-QOL, employment and disability as

reflected by lower SF-36 scores and employment rates, and higher disability rates when

compared with the general Dutch population. Moreover, analysis of HR-QOL revealed

that sSS patients were more limited in physical activities than pSS patients. Although the

results are obtained in a Dutch cohort of SS patients, the striking differences in HR-QOL,

employment and disability suggest that the results of our study are not limited to the Dutch

population, but probably are generally applicable to SS patients when compared with healthy

subjects.

Reduced HR-QOL in SS patients compared with normative data has been reported

before, but these studies were performed in smaller populations.(6;9;12;15) Overall, the

SF-36 scores for pSS patients in our study were comparable to those mentioned in earlier

literature. (8-10;15)

We observed more limitations in physical functioning in sSS than in pSS patients. This

is in contrast to the results described by Sutcliffe et al. (7) and Tensing et al.(9) The latter

studies were performed in smaller patient cohorts and mainly included sSS patients with

SLE as second autoimmune disease. The associated rheumatic disease in our sSS patients

was more diverse (RA, SLE and other). RA patients are considered to be more restricted

in physical functioning than SLE patients (16), which might explain the difference in results.

We, however, did not observe such a difference between sSS/RA and sSS/SLE patients;

perhaps because of the relatively small sSS subgroups in our study.

In our regression analyses several demographic and clinical factors were found to be

associated with HR-QOL. The unexplained variance probably reflects unmeasured, non-

disease related psychosocial factors such as self-esteem, support and coping strategies

(17), and other factors such as immunologic parameters, delay in diagnosis and untreated

or undiagnosed depression.(15) Interestingly, fatigue was an important explanatory variable

for reduced physical and mental HR-QOL. (5;9;18)This finding is in agreement with other

studies. Furthermore, the importance of fatigue in SS was underscored by the fact that the

majority of SS patients felt tired and 40% ranked fatigue as their most severe symptom.

Fatigue should therefore be considered as an important treatment target.

Segal et al.(19) demonstrated that psychological variables such as depression are

determinants for fatigue, but only partly account for it. Since depression could be of

importance for our outcome measures as well, the use of antidepressants was scored in

our population (9%). The regression analyses showed that antidepressants were a predictive

factor for mental HR-QOL, as can be expected; but not for physical HR-QOL, employment

or receiving DC.

We observed low employment and high disability rates in SS, which also have been

reported for rheumatic diseases such as RA (17;20) and ankylosing spondylitis.(17) To our

knowledge, these results have not previously been reported in SS patients.

A high level of education and comorbidity were the most significant predictors for having

paid employment. One would expect, however, that fatigue and arthralgia would also have

influenced the employment status. A possible explanation for the lack of this association could

be that, with time, patients have gradually adapted their activities to these symptoms. This

hypothesis is supported by the fact that only 10% of employed patients had a full-time job.

We found a higher frequency of EGM (95%) compared with other studies.(8;12;15) This

can partly be explained by the extended definition of EGM used in this study. Interestingly,

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26

Table 3 Linear multivariate regression analyses for the PCS and MCS of the SF-36.

PCS, model 1 PCS, adjusted for confounding

Variable ß [95% CI] P Variable ß [95% CI] P

Fatigue -24.26 [-33.07 – -15.44] 0.000 Fatigue -21.38 [-30.31 – -12.46] 0.000

Tendomyalgia -9.18 [-15.22 – -3.13] 0.003 Tendomyalgia -7.62 [-14.22 – -1.03] 0.024

Comorbidity -18.51 [-24.97 – -12.06] 0.000 Comorbidity -17.97 [-25.11 – -10.82] 0.000

Male sex

Receiving DC

-12.69 [-23.47 – -1.92]

-9.64 [-15.95 – -3.34]

0.021

0.003

Male sex

Receiving DC

Disease duration (years)

NSAID use

Antidepressant use

Employment

-11.38 [-22.11 – -0.65]

-10.71 [-17.13 – -4.29]

0.15 [-0.27 – 0.56]

-4.37 [-11.67 – 2.94]

-6.76 [-18.19 – 4.67]

-0.95 [-2.31 – 1.14]

0.038

0.001

0.487

0.239

0.244

0.217

MCS, model 1 MCS, adjusted for confounding

Variable ß [95% CI] P Variable ß [95% CI] P

Fatigue -15.97 [-24.48 – -7.45] 0.000 Fatigue -16.92 [-26.26 – -7.57] 0.000

Dry mouth 17.93 [5.94 – 29.91] 0.004 Dry mouth 16.75 [2.50 – 31.00] 0.022

Articular involvement -7.63 [-13.65 – -1.60] 0.008 Articular involvement -5.48 [-12.18 – 1.22] 0.108

Artificial saliva use

Antidepressant use

Comorbidity

-9.33 [-18.46 – -0.21]

-9.57 [-20.47 – 1.32]

-9.49 [-15.74 – -3.23]

0.045

0.085

0.003

Artificial saliva use

Antidepressant use

Comorbidity

Receiving DC

-12.58 [-22.97 – -2.20]-

-11.32 [-24.18 – 1.54]

-11.91 [-18.92 – -4.89]

-2.11 [-8.68 – 4.45]

0.018

0.084

0.001

0.526

PCS = physical component summary score; MCS = mental component summary score; ß = regressioncoeficient; 95% CI = 95% confidence interval; DC = disability

compensation; NSAIDs = non-steroidal anti-inf lammatory drugs.

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we found a higher frequency of Raynaud’s phenomenon (43%), as compared with the study

performed by García-Carrasco et al. (16%).(12) This may be explained by different weather

circumstances in The Netherlands. The observed higher prevalence of lymphoproliferative

disease (15% vs. 2%) may be related to the use of parotid gland biopsies in the diagnostic

work-up of our patients.(21) Parotid biopsies are more suited for (early) detection of

lymphoproliferative disease than labial biopsies as mucosa associated lymphoid tissue

(MALT) and non-Hodgkin lymphomas are rarely found in labial glands.

Although the percentage of patients with EGM did not differ between pSS and sSS

patients, it should be noted that part of the EGM in sSS patients could be attributed to

the associated autoimmune disease and not only to SS. EGM and EGM related treatment

were predictive for HR-QOL, employment and receiving DC. Therefore, there is a need for

accurate follow-up and treatment aimed at EGM.

The response rate of 83% in our study is very reasonable. As such, the risk of a sampling

bias of certain categories of patients to be preferentially included in this study is considered

negligible. Furthermore, we did not observe any significant differences for age, sex and

pSS/sSS ratio between responders and non-responders. We, therefore, conclude that our

results are representative for SS patients regularly attending a Medical Center specialized in

SS patient care.

Table 4 Education level, employment characteristics and disability compensation (DC) in SS patients of

working age.

Employment characteristics (n,%)

GDPn=135

SS patientsn=135

P SS patients vs GDP

pSS patientsn=109

sSS patientsn=26

P pSS vs sSS

Level of education

Low

Middle

High

Unknown

31 (23.5%)

57 (43.2%)

44 (33.3%)

5 (3.7%)

94 (69.6%)

33 (24.4%)

3 (2.2%)

<0.001 5 (3.8%)

75 (57.7%)

26 (20.0%)

3 (2.3%)

0

19 (57.6%)

7 (21.2%)

0

0.800

Paid employment 109 (82.6%) 69 (51.1%) <0.001 58 (53.2%) 11 (42.3%) 0.297

Full time paid job 26 (23.9%) 7 (10.1%) <0.01 7 (12.1%) 0 0.237

Hours worked per week

(mean±SD)26.9±14.2 21.7±11.6 0.011 21.7±12.1 21.3±8.5 0.914

Days sick leave per year

(mean±SD) NA 15.6±39.0 NA 14.7±37.8 22.3±50.0 0.675

Receiving DC 2 (1.5%) 63 (46.7%) <0.001 49 (45.0%) 14 (53.8%) 0.267

Full DC NA 28 (44.4%) NA 21(42.9%) 7 (50.0%) 0.434

Disability percentage

(mean±SD)NA 66.2±30.2 NA 63.6±30.0 75.8±30.0 0.246

Cause receiving DC

pSS, sSS or associated

rheumatic disease

Other

Unknown

NA 41 (65.1%)

7 (11.1%)

15 (23.8%)

NA 33 (67.3%)

6 (12.2%)

10 (20.4%)

8 (57.1 %)

1(7.1%)

5 (35.7%)

GDP = general Dutch population; n = number of patients; DC = disability compensation; NA = not avail-

able.

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Since many SS patients suffer from reduced HR-QOL and are restricted in social and

work related activities, there is a great need for developing adequate treatment modalities

to reduce SS related complaints and to intervene in the progression of SS. Currently, no

causal systemic treatment is available in SS and, therefore, only symptomatic treatment

can be given. Recently, some studies reported good results of treatment with biologicals,

especially anti-CD20 treatment.(22-25) Therefore, further development and evaluation of

systemic treatment options should be stimulated.

Table 5 Logistic multivariate regression analyses for employment and receiving disability compensation (DC) in

SS patients.

Employment, model 1 Employment, adjusted for confounding

Variable Odds ratio [95% CI]

P Variable Odds ratio [95% CI]

P

Bladder involvement 0.19 [0.05 – 0.75] 0.017 Bladder involvement 0.20 [0.05 – 0.81] 0.024

Oral moisturising gel use 0.32 [0.11 – 0.94] 0.038 Oral moisturising gel use 0.37 [0.12 – 1.15] 0.084

NSAID use 0.30 [0.12 – 0.81] 0.017 NSAID use 0.25 [0.09 – 0.70] 0.008

Oral corticosteroids use 0.16 [0.04 – 0.59] 0.006 Oral corticosteroids use 0.14 [0.04 – 0.56] 0.005

Comorbidity

Age at diagnosis (years)

0.13 [0.05 – 0.36]

0.95 [0.92 – 0.97 ]

0.000

0.000

Comorbidity

Age at diagnosis

0.14 [0.05 – 0.39]

0.97 [0.92 – 1.02]

0.000

0.261

High level of education 4.39 [1.69 – 11.44] 0.002 High level of education

Autoimmune thyroiditis

Artificial tears use

Age

4.21 [1.59 – 11.16]

0.46 [0.09 – 2.54]

0.50 [0.18 – 1.37]

0.97 [0.92 – 1.02]

0.004

0.376

0.177

0.250

Receiving DC, model 1 Receiving DC, adjusted for confounding

Variable Odds ratio [95% CI ]

P Variable Odds ratio [95% CI ]

P

Number of EGM 1.37 [1.04 – 1.80] 0.026 Number of EGM 1.28 [0.96 – 1.70] 0.099

Artificial saliva use 6.89 [1.92 – 24.76] 0.003 Artificial saliva use 6.21 [1.66 – 23.18] 0.007

Antimalarial drug use 3.41[1.19 – 9.74] 0.022 Antimalarial drug use 2.79 [0.94 – 8.32] 0.065

Comorbidity 2.70 [1.08 – 6.79] 0.034 Comorbidity 2.73 [1.05 – 7.11] 0.039

Age at diagnosis (years) 0.93 [0.90 – 0.97] 0.000 Age at diagnosis (years) 0.94 [0.90 – 0.97] 0.000

Male sex 23.11 [4.40 – 121.24] 0.000 Male sex 32.21 [5.23- 198.42] 0.000

High level of education 2.86 [1.09 – 7.50] 0.032 High level of educationFatigue

2.66 [1.00 – 7.06]

3.33 [0.67 – 16.57]

0.050

0.142

Skin involvement other than cutaneous vasculitis

1.35 [0.41 – 4.42] 0.625

Pilocarpine use 2.72 [0.76 – 9.74] 0.124

95% CI = 95% confidence interval; UTI = urinary tract infections; NSAIDs = non-steroidal anti-inf lam-

matory drugs; EGM = extraglandular manifestations.

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Conclusion

SS has a large impact on HR-QOL, employment and disability as reflected by lower SF-36

scores and employment rates, and higher disability rates in SS patients as compared with the

general Dutch population. Several demographical and clinical factors were associated with

HR-QOL, employment and receiving disability compensation. Physical functioning, bodily

pain and general health were worse in sSS than in pSS patients.

Acknowledgements

We would like to thank Dr. M. Pompen and Dr. E. Ten Vergert for their expertise in

the development of the questionnaire and Dr. M. Jalving for reading the manuscript and

providing constructive criticism. Also we would like to thank Prof. N.K. Aaronson and Mr.

C.M. Gundy of the Netherlands Cancer Institute and the Dutch Office of Statistics, for

providing us with age and sex matched normative data on HR-QOL, employment and DC.

For their assistance in analysing the data, we are gratefully to J. Bulthuis-Kuiper and R.P.E.

Pollard.

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(13) Ramos-Casals M, Font J, Garcia-Carrasco M, Brito MP, Rosas J, Calvo-Alen J et al. Primary Sjögren’s

syndrome: hematologic patterns of disease expression. Medicine (Baltimore) 2002; 81(4):281-92.

(14) Aaronson NK, Muller M, Cohen PD, Essink-Bot ML, Fekkes M, Sanderman R et al. Translation,

validation, and norming of the Dutch language version of the SF-36 Health Survey in community

and chronic disease populations. J Clin Epidemiol 1998; 51(11):1055-68.

(15) Belenguer R, Ramos-Casals M, Brito-Zeron P, del Pino J, Sentis J, Aguilo S et al. Influence of clinical

and immunological parameters on the health-related quality of life of patients with primary

Sjögren’s syndrome. Clin Exp Rheumatol 2005; 23(3):351-6.

(16) Benitha R, Tikly M. Functional disability and health-related quality of life in South Africans with

rheumatoid arthritis and systemic lupus erythematosus. Clin Rheumatol 2007; 26(1):24-9.

(17) Chorus AM, Miedema HS, Boonen A, Van Der Linden S. Quality of life and work in patients with

rheumatoid arthritis and ankylosing spondylitis of working age. Ann Rheum Dis 2003; 62(12):1178-84.

(18) Barendregt PJ, Visser MR, Smets EM, Tulen JH, van den Meiracker AH, Boomsma F et al. Fatigue in

primary Sjögren’s syndrome. Ann Rheum Dis 1998; 57(5):291-5.

(19) Segal B, Thomas W, Rogers T, Leon JM, Hughes P, Patel D et al. Prevalence, severity, and predictors

of fatigue in subjects with primary Sjögren’s syndrome. Arthritis Rheum 2008; 59(12):1780-7.

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of li

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(20) Verstappen SM, Boonen A, Bijlsma JW, Buskens E, Verkleij H, Schenk Y et al. Working status among

Dutch patients with rheumatoid arthritis: work disability and working conditions. Rheumatology

(Oxford) 2005; 44(2):202-6.

(21) Pijpe J, Kalk WWI, van der Wal JE, Vissink A, Kluin PM, Roodenburg JLN et al. Parotid gland

biopsy compared with labial biopsy in the diagnosis of patients with primary Sjögren’s syndrome.

Rheumatology (Oxford) 2007; 46(2):335-41.

(22) Devauchelle-Pensec V, Pennec Y, Morvan J, Pers JO, Daridon C, Jousse-Joulin S et al. Improvement

of Sjögren’s syndrome after two infusions of rituximab (anti-CD20). Arthritis Rheum 2007;

57(2):310-7.

(23) Meijer JM, Pijpe J, Bootsma H, Vissink A, Kallenberg CG. The future of biologic agents in the

treatment of Sjögren’s syndrome. Clin Rev Allergy Immunol 2007; 32(3):292-7.

(24) Pijpe J, van Imhoff GW, Vissink A, van der Wal JE, Kluin PM, Spijkervet FK et al. Changes in salivary

gland immunohistology and function after rituximab monotherapy in a patient with Sjögren’s

syndrome and associated MALT lymphoma. Ann Rheum Dis 2005; 64(6):958-60.

(25) Pijpe J, van Imhoff GW, Spijkervet FKL, Roodenburg JLN, Wolbink GJ, Mansour K et al. Rituximab

treatment in patients with primary Sjögren’s syndrome: An open-label phase II study. Arthritis

Rheum 2005; 52(9):2740-50.

Jiska M Meijer1, Justin Pijpe1, Hendrika Bootsma2, Arjan

Vissink1, Cees GM Kallenberg2

Clin Rev Allergy Immunol. 2007 Jun;32(3): 292-7

Chapter 3

The future of biologic agents in the

treatment of Sjögren’s syndrome

Departments of 1Oral and Maxillofacial Surgery, 2Rheumatology and Clinical Immunology,

University Medical Center Groningen, University of Groningen, The Netherlands

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Abstract

The gain in knowledge regarding the cellular mechanisms of T and B lymphocyte activity

in the pathogenesis of Sjögren’s syndrome (SS) and the current availability of various

biological agents (anti-TNF-α, IFN-α, anti-CD20, and anti-CD22) have resulted in new

strategies for therapeutic intervention. In SS, various phase I and II studies have been

performed to evaluate these new strategies. Currently, B cell-directed therapies seem to

be more promising than T cell-related therapies. However, large, randomized, placebo-

controlled clinical trials are needed to confirm the promising results of these early studies.

When performing these trials, special attention has to be paid to prevent the occasional

occurrence of the severe side effects.

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Introduction

Sjögren’s syndrome (SS) is a chronic lymphoproliferative autoimmune disease with

disturbances of T lymphocytes, B lymphocytes and exocrine glandular cells.(1) SS can be

primary (pSS) or secondary SS (sSS), the latter being associated with another autoimmune

disease (e.g. rheumatoid arthritis, systemic lupus erythematosus (SLE)).

Lymphocytic infiltrates are a characteristic histopathological finding in SS. These infiltrates

consist of T and B cells. The expression of different cytokines, such as tumor necrosis

factor-α (TNF-α) and interferon-α (IFN-α), during the formation and proliferation of these

infiltrates has been investigated. There is an over expression of TNF-α, which is secreted

by CD4+ T lymphocytes, mononuclear cells and epithelial cells.(2) The intraglandular

synthesis of TNF-α causes destruction of acini by up-regulation of Fas at the surface of the

glandular epithelial cells, stimulation of secretion of type 2 and 9 matrix metalloproteases

by epithelial cells, and over expression of different chemokines.(3-5) IFN-α is produced by

activated plasmacytoid dendritic cells in primary SS (pSS) and numerous IFN-α producing

cells have been detected in labial salivary glands.(6) IFN-α promotes the autoimmune

process by increasing autoantibody production and through the formation of endogenous

IFN-α inducers. IFNs have potent immunomodulating properties and are thought to trigger

a systemic biological response.(7)

Besides the presence of proinflammatory cytokines, described in the previous paragraph,

recent studies have shown an important role for B cells in the pathogenesis of SS. Presence

of autoantibodies and hypergammaglobulinemia are both considered to reflect B cell

hyperactivity. Systemic complications of SS are associated with this B cell hyperactivity.(8)

Moreover, about 5 % of SS patients develop malignant B cell lymphoma.(9) B cell activating

factor (BAFF), also known as B lymphocyte stimulator (BLyS), is an important factor in

local and systemic autoimmunity.(1) Dysregulated BAFF expression is implicated in disease

progression and perpetuation of humoral autoimmunity. Overproduction of BAFF in

transgenic mice has been shown to result in B cell proliferation and antibody production

resulting in inflammation and destruction of the salivary glands, as well as kidney failure

similar to observations seen in SLE.(10) In humans, circulating BAFF levels are increased in

patients with pSS and correlate with disease activity.(11)

Recent insights in the cellular mechanisms of T and B lymphocyte activity in the

pathogenesis of SS and the current availability of various biological agents have resulted

in new strategies for therapeutic intervention. The use of these biological agents in the

treatment of SS will be discussed in this review.

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Biological agents

Currently, biological agents have been introduced in various systemic autoimmune diseases,

as rheumatoid arthritis and SLE. Biological agents most frequently applied in autoimmune

diseases are monoclonal antibodies, soluble receptors and molecular imitators.(12) These

biological agents enhance or replace conventional immunosuppressive therapy. In contrast to

rheumatoid arthritis and SLE, no biological agent has been approved yet for the treatment of

SS, but several phase II and III studies have been or are currently conducted. The biological

agents used in SS trials are IFN-α and agents targeting TNF-α and B cells (anti-CD20, anti-

CD22). Although no trials have yet been performed with BAFF antagonists, this might be a

promising therapy(13) and will be discussed in this review, as well.

Anti-TNF-α monoclonal antibodies

There are three main biological agents targeting TNF-α : the chimeric monoclonal IgG1

antibody infliximab, the receptor fusion protein etanercept, and the fully humanized

monoclonal antibody adalimumab.

In an open-label study, short-term treatment with infliximab was reported to be very

effective in active pSS over a 3-month period.(14) Sixteen patients received 3 infusions (3mg/

kg) at weeks 0, 2 and 6, which led to significant improvement in all clinical and functional

parameters, including global assessments, erythrocyte sedimentation rate, whole salivary

flow rate, tear secretion (Schirmer test), tender joint count, fatigue score, and sensation

of dry eyes and dry mouth. Three patients, all with short disease duration (< 3 years),

were considered to be in complete remission up till 1 year. In 10 out of the 16 patients,

SS symptoms, particularly mouth dryness, relapsed after a median of 9 weeks. In a follow-

up study, a maintenance regimen of one infusion every 12 weeks was evaluated in these 10

patients. Retreatment induced an improvement of signs related to SS that was comparable

with the effects from the three loading infusions.(15) To confirm these promising results

from an uncontrolled study, the Trial of Remicade In Primary Sjögren’s Syndrome study was

designed. In this multicenter, double-blinded, placebo-controlled randomized clinical trial,

103 patients with active pSS were included and treated with infliximab infusions (5 mg/kg) or

placebo at weeks 0, 2 and 6. Follow-up was 22 weeks. Primary endpoint was an improvement

of >30% of two of three VAS scores measuring joint pain, fatigue and dry eyes. There were

several secondary endpoints of which one was the basal salivary flow rate. In contrast to the

previously mentioned uncontrolled studies, no evidence of efficacy of infliximab treatment on

all clinical and functional parameters could be demonstrated in this randomized controlled

clinical trial.(2)

A trial on 15 pSS patients (mean disease duration 3.6 years) with 25-mg etanercept,

subcutaneously twice a week for 12 weeks, did not reveal a reduction of sicca symptoms

and signs, neither did the repeated treatment for up to 26 weeks. Only in the subset of 4

patients with severe fatigue, a decrease of fatigue was observed.(16) Another trial evaluating

subcutaneous administration of etanercept versus placebo for 12 weeks (28 patients) also

showed no clinical efficacy.(17)

No trials of adalimumab treatment in pSS have been reported in the literature yet.

In conclusion, TNF-targeting treatment could not be proven to be of benefit in reducing

the complaints of pSS patients.

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IFN-αIFNs are proteins with antiviral activity and potent immunomodulating properties. SS

patients have an activated type I IFN system.(6) Such a role for IFN-α appears to contradict

the reports described below, that low doses of IFN-α administered via the oromucosal

route increase the unstimulated salivary output. However, it is hypothesized that oral IFN-α

treatment may act by increasing saliva secretion by upregulation of aquaporin 5 transcription

without significantly influencing the underlying autoimmune process.(6;7)

In a phase II study, treatment of pSS patients with IFN-α administered via the oromucosal

route (by dissolving lozenges) was demonstrated to be effective (improvement of salivary

output, decreased complaints of xerostomia) and safe.(18) Based on these promising results,

a randomized, parallel group, double-blinded, placebo-controlled clinical trial (497 pSS

patients) was designed. Patients were randomized into two groups and received a 24-week

daily treatment with either 450 IU IFN-α (150IU three times per day) or placebo in a ratio

3:2, administered by the oromucosal route. This randomized, controlled clinical trial failed

to demonstrate a significant effect on the primary endpoints (VAS score for oral dryness

and stimulated whole salivary flow) in the IFN-α group relative to the placebo group. There

was a significant increase in unstimulated whole saliva in the patients treated with IFN-α,

which correlated positively and significantly with improvement in seven of eight symptoms

associated with oral and ocular dryness. No adverse events were observed.(7)

In conclusion, no clinical evidence for the efficacy of IFN-α treatment in pSS patients

has been shown yet; however an improvement of unstimulated whole saliva was observed.

Further research is needed to objectify the effect of IFN-α on salivary gland tissue.

Anti-CD20 monoclonal antibodies

Anti-CD20 (rituximab) is a chimeric humanized monoclonal antibody specific for the B cell

surface molecule CD20, which is expressed on the surface of normal and malignant pre-B

and mature B lymphocytes. CD20 mediates B cell proliferation and differentiation. This

antibody has been demonstrated to prevent B cells from proliferating and to induce lysis of

B cells by complement-dependent and antibody-dependent cytotoxicity mechanisms as well

as by direct induction of apoptosis.(19)

Rituximab is currently used for the treatment of low-grade B cell lymphomas.(20) In

controlled studies, it was shown to be safe and effective in the treatment of rheumatoid

arthritis.(21-23) Moreover, open-label studies in SLE patients are promising.(24)

In an open-label phase II study, 15 patients with pSS were treated with 4 infusions of

rituximab (375 mg/m2 once weekly) and followed up for a 3-month period. Eight of the 15

patients were early pSS patients (mean disease duration 28 months, all had residual salivary

gland function at baseline), and 7 patients had a concomitant mucosa associated lymphoid

tissue (MALT) lymphoma (mean disease duration 79 months).

In the early pSS patients, rituximab treatment resulted in significant improvement of

subjective symptoms and an increase in salivary gland function. All patients showed a rapid

depletion of peripheral B cells within a few weeks, accompanied by a decrease in IgM-RF le-

vels.(8) Repeated parotid gland biopsies in five of the early patients after treatment, showed

redifferentation of the lymphoepithelial duct lesions into normal striated ducts, possibly

indicating regeneration of salivary gland tissue. (Unpublished data)

Five of the eight pSS patients without a MALT lymphoma received a second course of

rituximab (after 9-11 months) due to recurrence of symptoms. Retreatment resulted in the

same significant improvement of the salivary flow rate and subjective symptoms compared

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Table 1 Adverse events after treatment with biological agents in SS.

Agent/dose Number of patients in trial (number treated with the agent)

Premedication/ con-commitant immuno-suppressive therapy

Infusion reaction

Ìnfections Serum sickness

HACA / HAHA forma-tion

Other

Anti-TNF-α mono-clonal antibodies

Steinfeld(14) Infliximab Intravenous, 3 mg/kg

16 (16) n.r. /no 1 (6%) 2 (13%) (respiratoy tract)

- n.r. -

Steinfeld(15) InfliximabIntravenous, 3 mg/kg

10 (10) n.r. /no 4 (40%) 2 (20%) (enteritis, tonsillitis)

- n.r. -

Marriette(2) InfliximabIntravenous, 5 mg/kg

103 (54) n.r. /continuation of hydroxychloroquine and corticosteroids (≤ 15 mg/day)

2 (4%) 2 (4%) (1 cutane-ous, 1 respiraotry tract)

- n.r. 2 (breast cancer, auto-immune hepatitis) †

Zandbelt(16) Etanercept subcutaneously, 25 mg

15 (15) n.r. /pilocarpine at a constant dose

- 1 (7%) (parotitis) - n.r. -

Sankar(17) Etanercept subcutaneously, 25 mg

28 (14) n.r. /allowed to use long-term medication

1 (7%) 1 (7%) (skin le-sion) ‡

- n.r. -

IFN-αShip(18) IFN-α oromucosal, 150 IU,

450 IU109 (87) n.r. /no n.a. - - n.r. - ¶

Cummins(7) IFN-α oromucosal, 450 IU 497 (300) n.r. /no n.a. - - - 23 (7.7%) § (34% gastrointestinal, 25% musculoskeletal)

Anti-CD20Pijpe(8) Rituximab

Intravenous, 375 mg/m²15 (15) 25 mg prednisolon

intravenously/ patients with severe extraglandu-lar manifestations (n=3) received immunosup-pressive therapy

2 (13%) 1 (7%) (zoster) 4 (27%) # 4 (27%) -

Devauchelle-Pensec(25)

RituximabIntravenous, 375 mg/m²

16 (16) n.r. /no - - 1 (6%) n.r. -

Gottenberg(26) RituximabIntravenous, 375 mg/m²

6 (6) n.r. / hydroxychloroquine (n=1), methylpredniso-lone (n=3)

1 (17%) - 1 (17%) n.r. -

Seror(27) RituximabIntravenous, 375 mg/m²

12 (12) n.r. / cyclophosphamide (n=1), hydroxychloro-quine (n=1), leflunomide (n=1)

1 (8%) - 2 (17%) n.r. -

Anti-CD22

Steinfeld(29) EpratuzumabIntravenous, 360 mg/m²

16 (16) 0.5-1 g acetominophen, 25-50 mg antihistamine./no

2 (13%) 2 (13%) (sinusitis, dental abcess)

- 3 (19%) 6 (38%) (TIA, osteo-porotic fracture, diar-rhea, dyspepsia, palpita-tions, paresthesia)

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to the results of the first treatment, together with a decrease in B cells and IgM-RF levels.

Six of the seven MALT/pSS patients were initially effectively treated with rituximab. The

remaining MALT/pSS patient had progressive MALT disease and severe extraglandular SS

disease within three months after the start of rituximab treatment. Cyclophosphamide was

added, which led to stable disease of both MALT and SS. One of the six patients initially

responding had a recurrence of MALT lymphoma after 9 months and was successfully

retreated with rituximab. The other patients are still in remission. (Unpublished data)

In another open label study, 16 pSS patients received only two weekly rituximab infusions

(375 mg/m2), with a follow-up of 36 weeks. Again, treatment resulted in rapid complete

depletion of peripheral B cells. At week 12, a significant improvement of VAS scores for

fatigue and dryness was recorded, and at week 36, a significant improvement for VAS scores

for global disease, fatigue, dry mouth, dry eyes and dry vagina, but also in the number of

tender joint and tender point counts was seen.(25) Both in the study of Pijpe et al.(8) and

the study of Devauchelle-Pensec et al.(25) patients with a short disease duration showed

more improvement than patients with longer disease duration.

Two trials retrospectively evaluated the effect of rituximab (4 infusions of 375 mg/m2)

in 18 pSS patients (mean disease duration 10 years) with systemic features. Self-reported

dryness improved in six patients (VAS scores not known for three patients, no improvement

in the other nine patients). Both studies reported good efficacy of the treatment on

systemic features.(26;27)

In conclusion, in phase II trials, it has been shown that rituximab seems to be effective

for at least 6-9 months in pSS patients with active disease, improving both subjective

and objective complaints. Retreatment with rituximab resulted in a similar good clinical

response. In pSS patients with longer disease duration, without residual salivary gland

function, rituximab treatment seems to be effective for systemic features. To confirm these

promising results, randomized placebo-controlled clinical trials are needed.

Anti-CD22 monoclonal antibodies

Epratuzumab is a fully humanized monoclonal antibody specific for the B cell surface

molecule CD22. CD22 is expressed on the surface of normal mature and malignant B

lymphocytes. CD22 appears to be involved in the regulation of B cell activation through

B cell receptor signaling and cell adhesion.(28) In an open label phase I/II study, safety and

efficacy of epratuzumab were investigated in 16 pSS patients. Follow up was 6 months.

These pSS patients received four doses of 360 mg/m² epratuzumab intravenously. Mean

disease duration before therapy was 2.9 years, and none of the patients had received prior

B cell-targeted therapy. Most improvements occurred in the Schirmer test, unstimulated

† 1 patient in the placebo group developed benign lymph node enlargement

‡ 1 patient in the placebo group developed a prolonged upper respiratory tract infection

¶ In this study there were mild adverse events, however there were no significant differences

between the groups. Adverse events were not specified

§ 8 patients (4.1%) in the placebo group developed adverse events # 1 of these 4 patients developed serum sickness after retreatment (8)

n.a. not applicable

n.r. not reported

HACA human anti-chimeric antibodies

HAHA human anti-human antibodies

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whole salivary flow and the VAS score for fatigue. The new developed disease activity

score consisted of the four domains: dryness of the eyes, dryness of the mouth, fatigue

and laboratory parameters. Based on this score 53% achieved at least 20% improvement in

at least two domains at 6 weeks. Corresponding rates for 10, 18, and 32 weeks are 53, 47

and 67%. Remarkably, the number of responders was higher 6 months after the treatment

administration than earlier. Peripheral B cells decreased with a median decrease of 54 and

39% at 6 and 18 weeks, respectively.

In conclusion, epratuzumab seems to be an effective treatment. Randomized, placebo-

controlled clinical trials are needed before epratuzumab can be advised for general

treatment in pSS patients.(29)

Anti-BAFF

BAFF is a B cell-activating factor that acts as a positive regulator of B cell function and

expansion. BAFF levels were found elevated in serum and saliva in SS patients, but no

correlation could be shown between serum and saliva levels.(30) However, circulating levels

of BAFF in pSS patients were shown to be a marker for disease activity.(11)

To the best of our knowledge, no trials have been performed with anti-BAFF treatment

in SS yet, but such an approach might be considered for future trials. Currently, 2 human

BAFF antagonists have been developed, a human antibody (anti-BLyS) that binds to soluble

BAFF and a fusion protein of one of the BAFF receptors.(31;32) Especially SS patients with

elevated BAFF levels, hypergammaglobulinemia, elevated levels of auto antibodies, and

associated B cell lymphoma might be candidates for anti-BAFF treatment. (33)

Safety and tolerability of biological agents

The most important side effects of treatment with biological agents are direct mild infusion

reactions. Several patients developed a serum sickness-like disease a few days after the

second infusion that might be related to the formation of antibodies against the biological

agent (human anti-chimeric antibodies (HACA’s) or human anti-human antibodies). A few

patients developed infections during treatment with a biological agent; however, some

patients concomitantly used other immunosuppressive therapies. Therefore, the direct

relation between the biological agent and the infection is unsure. All adverse events

reported in the trials described in this review are reported in table 1. According to this

table, the most frequent side effects of treatment with biological agents are mild infusion

reactions. The most severe side effect of the various treatments used in SS patients was the

development of a serum sickness-like disease. This adverse effect of treatment occurred

in 16% (8 of 49) of the patients treated with rituximab. HACA formation was observed in

patients developing a serum sickness-like disease and occurred only in patients receiving

low-dose corticosteroids and no other immunosuppressive drugs. It is assumed that higher

doses of corticosteroids during treatment might prevent the occurrence of serum sickness.

Future perspectives

Biological agents are promising therapies for SS. Randomized studies failed to show a clinical

effect of anti-TNF-α and IFN-α in the treatment of SS. Notwithstanding the unfortunate

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results of anti-TNF-α and IFN-α, B cell depletion (both anti-CD20 and anti-CD22) seems

very promising. Again, this promising effect, as was previously also assumed for anti-TNF-α

and IFN-α, must be confirmed in larger randomized controlled clinical trials.

HACA’s have been reported to occur at a higher rate in patients with an autoimmune

disease. It seems that monoclonal antibodies are more immunogenic in active autoimmune

disease, independent of the type of disease. Additional use of immunosuppressive therapy

in these patients might be mandatory to prevent serious side effects. These unwanted side

effects might also be prevented by the use of fully humanized antibodies. The currently

available humanized antibodies are promising, but need further study. Moreover, there is still

a need for improved assessment parameters to monitor treatment effects, both subjectively

and objectively. For studies on intervention of SS, evaluation of the parotid gland might be

of use because function, composition of saliva and histology can be evaluated on the same

gland at different time-points. Activity scores are currently under development by Bowman

and Vitali.(34;35) Finally, as soon as effective intervention treatments have been established,

the cost-effectiveness of these currently very expensive antibodies needs to be analyzed to

select those patients that might benefit the most from this kind of treatment.

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of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med 2004;

350(25):2572-81.

(22) Edwards JC, Cambridge G. B-cell targeting in rheumatoid arthritis and other autoimmune diseases.

Nat Rev Immunol 2006; 6(5):394-403.

(23) Emery P, Fleischmann R, Filipowicz-Sosnowska A, Schechtman J, Szczepanski L, Kavanaugh

A et al. The efficacy and safety of rituximab in patients with active rheumatoid arthritis despite

methotrexate treatment: results of a phase IIB randomized, double-blind, placebo-controlled,

dose-ranging trial. Arthritis Rheum 2006; 54(5):1390-400.

(24) Looney RJ, Anolik JH, Campbell D, Felgar RE, Young F, Arend LJ et al. B cell depletion as a novel

treatment for systemic lupus erythematosus: a phase I/II dose-escalation trial of rituximab. Arthritis

Rheum 2004; 50(8):2580-9.

(25) Devauchelle-Pensec V, Pennec Y, Morvan J, Pers JO, Daridon C, Jousse S et al. Efficacy of rituximab

(anti-CD20) in the treatment of primary Sjögren’s syndrome (pSS): a 36 weeks follow-up. Arthritis

Care and Research. In press 2007.

(26) Gottenberg JE, Guillevin L, Lambotte O, Combe B, Allanore Y, Cantagrel A et al. Tolerance and short

term efficacy of rituximab in 43 patients with systemic autoimmune diseases. Ann Rheum Dis 2005;

64(6):913-20.

(27) Seror R, Sordet C, Gottenberg JE, Guillevin L, Masson C, Sibilia J et al. Good tolerance and efficacy

of rituximab on systemic features in 12 patients with primary Sjögren’s syndrome. Arthritis Rheum.

52[9 (supplement)]. 2005.

(28) Carnahan J, Wang P, Kendall R, Chen C, Hu S, Boone T et al. Epratuzumab, a humanized monoclonal

antibody targeting CD22: characterization of in vitro properties. Clin Cancer Res 2003; 9(10 Pt

2):3982S-90S.

(29) Steinfeld SD, Tant L, Burmester GR, Teoh NK, Wegener WA, Goldenberg DM et al. Epratuzumab

(humanized anti-CD22 antibody) in primary Sjogren’s syndrome: An open-label Phase I/II study.

Arthritis Res Ther 2006; 8(4):R129.

(30) Pers JO, d’Arbonneau F, Devauchelle-Pensec V, Saraux A, Pennec YL, Youinou P. Is periodontal

disease mediated by salivary BAFF in Sjögren’s syndrome? Arthritis Rheum 2005; 52(8):2411-4.

(31) Baker KP, Edwards BM, Main SH, Choi GH, Wager RE, Halpern WG et al. Generation and

characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities

of B lymphocyte stimulator. Arthritis Rheum 2003; 48(11):3253-65.

(32) Ramanujam M, Davidson A. The current status of targeting BAFF/BLyS for autoimmune diseases.

Arthritis Res Ther 2004; 6(5):197-202.

(33) Szodoray P, Jonsson R. The BAFF/APRIL system in systemic autoimmune diseases with a special

emphasis on Sjögren’s syndrome. Scand J Immunol 2005; 62(5):421-8.

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(34) Bowman SJ, Sutcliffe N, Price E, Isenberg D, Goldblatt F, Regan M et al. Measuring systemic disease

activity in primary Sjögren’s syndrome. Arthritis Rheum. 52, 376S. 2005.

(35) Vitali C, Palombi G, Baldini C, Benucci M, Bombardieri S, Covelli M et al. Measurement of disease

activity in Sjögren’s syndrome (sjs) by means of a new scale (sjsdam) developed by the analysis of a

cohort of patients collected by the study group for sjs of the italian society of rheumatology. Ann

Rheum Dis 65[suppl II], 606. 2006.

Review on biologicals45

Jiska M Meijer1, Cees GM Kallenberg2, Arjan Vissink1

In: Wong DT. Salivary diagnostics. Ames (IA): Wiley-Blackwell; 2008. 214-25

Chapter 4a

Progression and treatment evaluation in

diseases affecting salivary glands

1Departments of Oral and Maxillofacial Surgery, 2Rheumatology and Clinical Immunology,

University Medical Center Groningen, University of Groningen, The Netherlands

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Abstract

A general overview of existing tools for evaluation of treatment for diseases affecting salivary

glands is given. Assessments of salivary gland function (sialometry, sialochemistry) and

histopathological examination of salivary gland biopsies provide powerful tools to diagnose

diseases affecting the salivary glands, to assess disease progression and to evaluate treatment.

More general tools are subjective questionnaires (e.g. visual analogue scale (VAS) scores,

Multidimensional Fatigue Inventory (MFI) score and SF-36) and serological parameters.

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Introduction

Many diseases and conditions can affect salivary glands resulting in a reduced or increased

salivary flow. Treatment for these and other disorders can affect salivary secretion as well.

Frequent causes of long-lasting reduced salivary flow are drugs, systemic conditions like

Sjögren’s syndrome (SS) and radiation injury to salivary gland tissue. The sensation of a

dry mouth (xerostomia) is not always accompanied by a reduced salivary secretion (hypo-

salivation). In about one third of the patients with xerostomia there is no good correlation

between actual mouth dryness and level of salivary secretion. The discrepancy between

salivary secretion status and level of complaints is even more striking in drooling. Usually,

salivary secretion is normal or even reduced, but swallowing of saliva is impaired. Well-

known causes of the inability to empty the mouth of saliva are an infantile swallowing

pattern, a disturbed sensibility of the oral tissues and anatomic limitations due to trauma

and ablative surgery. Thus, many factors have to be considered when selecting a salivary

evaluation tool for the subset of patients or healthy subjects.

Notwithstanding the above, salivary research provides powerful tools to diagnose

diseases affecting salivary glands, to assess disease progression, and to evaluate treatment.

In progressive diseases like SS, salivary secretion generally diminishes with time. (figure 1)

This progression is not so obvious when monitoring whole saliva, but becomes much clearer

when measuring gland specific saliva.(1) While sialometry is a robust tool for evaluating

disease progression, analysis of salivary composition (sialochemistry) differentiates between

salivary gland diseases, and measures the disease activity (table 1)(2) and the effect of

intervention treatment.(3) Additional tools are sialography (imaging of the extent of

destruction of the ductal system), salivary scintigraphy (imaging of the glandular secretory

activity), salivary gland biopsy (glandular pathology underlying the observed changes), and

the imaging of anatomical structures with CT, MRI, or ultrasound.

The six above-mentioned variables (sialometry, sialochemistry, sialography, salivary

scintigraphy, biopsy, and imaging) are gland-specific and measure disease progression

and/or activity. Other essential information might come from the pattern of complaints,

medical history, the clinical picture, serology and questionnaires. Serological parameters

and subjective questionnaire responses can add important information on the disease

progression and treatment outcome.

This chapter discusses the main tools for evaluation of disease progression and treatment

including applications to clinical research and practice.

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Tools to measure salivary gland function and disease activity

Sialometry

Saliva collection provides sound clinical information. Accurate measures of salivary flow rate

and composition are essential for many diagnostic, therapeutic and research protocols. Saliva

collection is a noninvasive tool of assessing a variety of disease characteristics and levels of

certain drugs and hormones. Whole saliva is a mixture of not only salivary secretions, but

also fluids, debris, and cells not originating in the salivary glands. Therefore, the analysis of

individual gland saliva is usually a more reliable procedure for diagnosing diseases of the

salivary glands than analysis of whole saliva. However, for certain diagnostic procedures

whole saliva might be more useful, for example, when assessing specific roles of saliva in the

oral cavity or when whole saliva is used as a diagnostic fluid for conditions relying on leakage

of serum products or gingival crevicular fluid into saliva.

In healthy subjects and patients in whom both glands are affected simultaneously (e.g. SS)

flow rates of the left and right parotid gland are similar. Therefore, sorting out discrepancies

between the observed flow of the left and right parotid gland assures the reliability of the

samples collected. This is a very powerful internal control of the reliability of the saliva sample

collected and outweighs the effect of repeated sampling of a parotid gland to get a reliable

baseline sample. Increasing the number of collections has been shown to have a negligible

effect on the reliability of baseline parotid flow rates for clinical trials. Consequently, one

reliable baseline sample is sufficient for clinical studies evaluating the progression of disease

or the effect of a therapy.(4) Moreover, salivary flow rates are not constant and exhibit a

considerable amount of variability. Therefore, salivary collections should be performed under

well-defined conditions and, for repeated collections, at the same time of the day to minimize

intrapatient variability. Nevertheless, even if the circadian rhythm is ruled out and the samples

are indeed collected under well-defined conditions, the measured increase or decrease of

salivary flow has to exceed about one-quarter to one-third of the parotid flow rate at baseline

before an observed effect related to a given therapy can be assessed as a ‘real’ effect in an

individual patient. This information is additional to subjective assessments of such an effect.(4)

Sialochemistry

Saliva is an attractive diagnostic fluid because salivary testing provides several key advantages

including low cost, noninvasiveness, and easy sample collection and processing. Human saliva

collection is less invasive than phlebotomy and is clinically relevant because many, if not all,

blood components are reflected in saliva. Amongst others, sodium, potassium, chloride,

calcium, phosphate, urea, total protein and a number of enzymes (e.g., amylase, lysozyme

and lactoferin) can be detected in saliva and have diagnostic potential. (table 1) In addition,

a large range of more or less disease-related changes in protein composition of saliva have

been reported. A new method to assess the protein composition in health and disease is

salivary proteonomics - the identification of the entire spectrum of proteins in human saliva.

Saliva also harbours diagnostic RNA biomarkers (detection of RNA biomarkers).

Sialography

Through retrograde infusion of oil- or water-based iodine contrast, the architecture of

the salivary duct system is visualized radiographically. It is a low morbidity, well-accepted

technique. Sialography should not, however, be performed in patients with a history of

iodine allergy. The sialographic procedure can be performed in 10 -15 minutes.

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Figure 1

Relationship between disease duration (time from first complaints induced by or related to oral dryness

until referral) and mean (SEM) salivary flow rates in patients with (A) primary SS(pSS) and in those with (B)

secondary SS(sSS). Normal values are derived from historic controls (n=36). SM/SL, submandibular/sublin-

gual glands; UWS, unstimulated whole saliva. *Significant difference versus patients with early-onset SS(<

1 year oral complaints; p<0.005) by the Mann-Whitney U test. †Significant difference versus patients with

early-onset SS(p<0.05) by the Mann-Whitney U test (Pijpe et al. (1), reprinted with permission).

Inflammation appears on sialograms as diffuse collections of contrast fluid at the terminal

acini of the ductal tree. This condition, known as sialectasia can be classified into punctate

(less than 1 mm), globular (uniform and 1-2 mm), cavitary (coalescent and >2 mm) and

destructive (normal ductal structures are no longer visible). Sialectasia is thought to result

from progressive acinar atrophy and dilatation, which, in turn, is caused by increasing

intraluminal pressure resulting from the presence of periductal lymphocytic infiltrates with

secondary duct narrowing. So, these four grades of sialectasia are thought to represent

increasing glandular damage, caused by chronic salivary gland inflammation.

Chapter 4a

52

Table 1 Salivary gland parameters and clinical data of some disorders affecting the salivary glands (Van den Berg et al., 2007) (2). SS is an autoimmune disorder

affecting the exocrine glands including the salivary glands. Sialosis is a salivary condition characterized by persistent swelling of the parotid glands related to a

metabolic disorder as diabetes, alcohol abuse, anorexia and bulimia. Sodium retention syndrome is characterized by mostly unilateral, incidental, short-lasting (hours)

swelling of the parotid gland often related to cardiovascular disorders (hypotension, hypertension).

SS (pSS/sSS) Sialosis Sodium retention syndrome Medication induced xerostomia

Sialometry UWS ≤1.5 ml in 15 min Normal, increased or decreased Normal or decreased UWS decreased; SWS (sub) normal

Sialochemisty Na and Cl increased K increased Na decreased Normal

Sialography Sialectasia Thin duct system, enlarged gland Usually normal, but a thin duct system and enlarged gland may be present

Normal

Complaints Mouth dryness in rest and during eat-ing or speaking Need for drinks to swallow (dry) foodEye drynessSwelling of the salivary glands

Persistent, bilateral swelling of the parotid glands

Often mouth dryness. Recurrent, short lasting (usually at most some hours), mostly unilateral swellings of the parotid gland

Mouth dryness in rest

Schirmer’s test ≤5mm/5min Unknown, but reduction is not uncommon

Unknown, but reduction is not uncommon

Unknown, but reduction is not uncommon

AssociatedDiseases

sSS: associated with another connec-tive tissue/auto-immune disease

Endocrine disorder Metabolic disorder Dysfunction ANS

Cardiovascular diseaseDisorder of the fluid or electrolyte balance

Use of xerogenic medication

SS: Sjögren’s syndrome; pSS: primary Sjögren’s syndrome; sSS: secondary Sjögren’s syndrome; UWS: unstimulated whole saliva; SWS: stimulated whole saliva;

SM/SL: saliva from sublingual/-mandibular gland; ANS: autonomic nervous system.

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Salivary scintigraphy

Salivary scintigraphy is based on the ability of parotid and submandibular glands to trap the

radionuclide isotope technetium-sodium (Tc99m) pertechnetate. This ability is due to the

fact that Tc99m substitutes for chloride in the active sodium/potassium/chloride cotransport

in the striated ducts. After intravenous injection of Tc99m, scintigraphy may reveal functional

abnormality of the salivary glands through photographically recording with a gamma

scintillation camera, the radiation from salivary isotope accumulation and excretion.

Improvements of salivary scintigraphy include salivary single-photon emission computed

tomography (SPECT) and human immunoglobulin G (HIG) scintigraphy. Salivary SPECT

creates a three-dimensional image with a rotating gamma camera without marking an ROI

(region of interest) as it uses a single pixel as the ultimate ROI.

Scintigraphy is a valuable tool to measure activity of the glands, and it can be performed

in the same gland at different time periods to assess progression. Unfortunately, the

diagnostic accuracy is low.

Computer tomography and magnetic resonance imaging

Magnetic resonance imaging (MRI) depicts more accurately because soft tissue contrast

resolution is better in MRI than computer tomography (CT). Detailed knowledge of

the anatomy of the parotid gland and surrounding structures is necessary for evaluating

and diagnosing lesions. Bilateral imaging and comparison between right and left glands is

essential. CT and MRI are of less value as diagnostic tools for salivary gland disorders as

Sjögren’s syndrome, sialadenosis and bacterial or viral sialadenitis.

Ultrasound

Ultrasound has no known contraindications and is a quick and well-accepted, non-invasive

procedure. With color Doppler sonography, the complex vascular anatomy can be

Figure 2

Flow rate of parotid and submandibular/sublingual saliva (SM/SL) as a function of time after start of radio-

therapy (conventional fractionation schedule, 2Gy per day, 5 days per week, total dose 60-70 GY). The

parotid, submandibular and sublingual glands are located in the treatment portal. Initial flow rates were set

to 100% (Adapted from Burlage et al. (12)).

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accurately recorded.(5) Its potential in routine salivary diagnostics is restricted as tissue

penetration depth is limited and proper interpretation of salivary sonograms requires a

great deal of experience.

Histopathology

The labial and parotid glands are accessible for histopathological evaluation, and biopsies from

these glands are often performed routinely. In SS, a disease affecting the salivary glands in which

biopsies most often are taken as a routine procedure, the parotid and labial gland biopsies are

diagnostically comparable. However, a parotid biopsy is preferred, due to lower morbidity than

labial biopsies in which sensory loss may occur, easier access to larger tissue samples, and earlier

detection of lymphomas.(6) In addition, repeated biopsies can be taken from the same parotid

gland, making parotid biopsies an important tool in treatment evaluation (the outcomes can even

be compared with saliva samples obtained from the same gland).

Cytology

A cytological puncture (ultrasound guided) can distinguish salivary gland disorders from

lymph nodes disorders, and inflammation from malignancy.

  ● Stimulated submandibu-

lar/sublingual salivary

flow rate

   ♦ IgM-RF

∗ B cells

▲ VAS score for dry mouth

during the night

▼ MFI score for fatigue

Figure 3

Increase and decrease (mean values of 5 patients) in stimulated submandibular/sublingual flow rate, IgM-RF,

B cells, VAS score for dry mouth during the night and multidimensional fatigue (MFI) score for fatigue fol-

lowing rituximab (re)treatment (baseline is 100%). Baseline values (week 0 first treatment) were stimulated

submandibular/sublingual flow rate 0.09 ml/min (SD 0.07), IgM-RF 339 (SD 329), B cells 0.19 109/l (SD

0.09), VAS score for dry mouth during the night 85 (SD 12), MFI score for fatigue 16 (SD 3). (modified after

Meijer et al.(13)).

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Subjective evaluation

VAS

A Visual Analogue Scale (VAS) is a line of, for example, 100 mm on which the patient can

mark the severity of the complaint. For SS, VAS scores are available for oral dryness, oral

dryness during the day, oral dryness at night, difficulty swallowing dry food without any

additional liquids, difficulty swallowing any food without any additional liquids, difficulty

speaking without drinking liquids, dry eyes (sensation of sand or gravel in the eyes).

MFI

The Multidimensional Fatigue Index (MFI) is a 20-item self-report instrument designed to

objectively measure fatigue, including the dimensions of general fatigue, physical fatigue,

mental fatigue, reduced motivation and reduced activity. This validated questionnaire detects

expected differences in fatigue between groups, within groups and between conditions.

(7) A higher score (range 4-20) indicates a higher level of fatigue. Fatigue is a complaint

not uncommon to patients suffering from salivary gland disorders, particularly patients

with salivary gland disorders related to an autoimmune disease or as a result of cancer

treatment.

SF-36

The 36-item short form (SF-36) is constructed to survey health status and was designed

for use in clinical practice and research, health policy evaluations and, general population

surveys. The SF-36 includes one multi-item scale that assesses eight health concepts. The

questionnaire has been developed for self-administration by persons 14 years of age and

older or for administration by a trained interviewer. A higher score indicates a higher

level of well-being.(8) Health status can severely be impaired in patients suffering from

salivary gland disorders particularly in patients with salivary gland disorders related to an

autoimmune disease or as a result of cancer treatment.

Dry mouth questionnaires

Objective salivary gland function is not always consistent with the subjective perception.

Whether the patient reports sipping liquids to aid in swallowing dry foods, dry mouth

when eating a meal, or difficulties swallowing any foods is highly predictive of salivary gland

function and, therefore, clinically useful in patients who report oral dryness.(9)

Serological parameters

In systemic diseases affecting the salivary glands, serological parameters can be useful

in evaluating activity and progression of the disease and in evaluating treatment. For

example, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) are

general parameters in peripheral blood for inf lammation and are elevated in most

autoimmune diseases. IgM-Rf (rheumatoid factor) and IgA correlate with B cell

activity and are elevated in SS. IgM-Rf is also elevated in patients with rheumatoid

arthritis and some other conditions. Antinuclear antibodies (ANA) and anti-Ro/

SSA and anti-La /SSB can be detected in SS (anti-SSB is the most specif ic antibody).

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Application of these tools in clinical research and clinical practice

The clinical application of the above-mentioned variables in treatment evaluation will be

illustrated for patients with a reduced salivary flow due to head and neck radiotherapy and

SS.

Radiotherapy

Xerostomia is a common and disturbing side effect of head and neck radiotherapy, leading to

considerable morbidity, including severe oral discomfort, problems with speaking, dysphagia,

and an increased incidence of caries and mucosal infections. Although new radiation

techniques enabled significant sparing of the parotid glands, the amount of normal salivary

gland tissue irradiated may still be substantial resulting in clinically relevant radiation-induced

xerostomia. Therefore, protection against radiation-induced salivary gland damage may

further improve the therapeutic gain.(10;11)

Although salivary gland tissue is a well-differentiated tissue and, theoretically, should be

relatively radioresistant, studies have shown a rapid decline in parotid and submandibular/

sublingual salivary flow, even after low doses of radiotherapy (figure 2). In humans, it has

been reported that the TD50

(i.e., the dose to the whole organ leading to a complication

probability of 50%) for parotid glands varies from 28.4 Gy to 31 Gy at 6 weeks increasing to

39 Gy at 1 year after completion of radiotherapy.

Sjögren’s syndrome

SS is a chronic lymphoproliferative autoimmune disease with disturbances of T lymphocytes,

B lymphocytes and exocrine glandular cells. SS can be primary (pSS) or secondary (sSS),

the latter being associated with another autoimmune disease (e.g. rheumatoid arthritis

and systemic lupus erythematosus). The main symptoms of SS are xerostomia, dry eyes

(keratoconjunctivitis sicca), increased caries activity (exocrine glands) and fatigue and

arthralgia (systemic features). The disease can have a great impact on the quality of life of

the patients. There are no causal treatment options, and treatment used today is mainly

symptomatic. Dry eyes are treated with eyedrops or gel, and sometimes anti-inflammatory

or immunosuppressive medication is indicated. Dry mouth is treated with saliva-stimulating

medication (pilocarpine) or with saliva substitutes. Currently, drug trials are evaluating

biological agents with promising first results. (figure 3)

Sialometry and sialochemistry

Salivary flow rates have diagnostic and prognostic value in SS. Since the amount and

composition of saliva reflects the effects of the autoimmune process in the salivary glands,

analysis of saliva may also be valuable in diagnosis, prognosis and evaluation of treatment.

SS is characterized by a high sodium and high chloride concentration and a low phosphate

concentration in parotid gland saliva.

Sialometry and sialochemistry, easily performed and tolerated, are valuable in measuring

disease progression (figure 1) and treatment outcome. For example, rituximab significantly

increased salivary secretion (figure 3) and nearly normalized salivary sodium concentration.

A pilot study of ten SS patients and ten age- and sex-matched controls demonstrated

that pSS patients’ saliva contains proteomic and genomic diagnostic biomarker candidates.

Proteonomics of saliva may also be useful in diagnosis, disease progression, and treatment

evaluation, but further research is necessary to precisely assess its value.

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Histopathology

In SS, widely accepted criteria for histologic confirmation is focal lymphocytic sialoadenitis

in labial salivary glands and lymphoepithelial lesions in parotid salivary glands.

Moreover, repeated salivary gland biopsies might offer an objective method for evaluating

treatment, in addition to serological and functional parameters. The parotid gland is the

primary site to study changes after systemic therapy since SS lymphoproliferation occurs

especially in these glands. Repeated parotid biopsies in SS patients treated with rituximab

show redifferentation of lymphoepithelial lesions into regular ducts, which is in line with the

sialochemical changes in parotid saliva.

Subjective evaluation

Fatigue is one of the most disabling complaints in SS, and it leads to a substantial decrease

in health related quality of life. By using the MFI, patients with pSS reported more fatigue

than healthy controls on all the dimensions of the MFI, and when controlling for depression

significant differences remain on the dimensions of general fatigue, physical fatigue, and

reduced activity. VAS scores have been used to assess subjective sicca complaints and have

been validated for patients with xerostomia. After rituximab treatment, in patients with

early pSS, assessment of mouth dryness, arthralgia, physical functioning, vitality and most

domains of the MFI significantly improved.(3)

Serological parameters

Polyclonal expansion and secretory hyperactivity of B cells is an early event in pSS. This

is demonstrated in the blood by increased amounts of different autoantibodies and by

increased amounts of total Ig (primarily IgG). The more serious systemic complications

occur mainly in patients with increased IgM-Rf levels, and levels of circulating IgM-Rf

correlate positively with the number of extraglandular disease manifestations. Other

researchers also reported an association between a high B cell autoreactivity (production of

ANA, anti-Ro/SSA and anti-La/SSB) and the development of complications or more severe

manifestations like neuropathy, kidney and pulmonary involvement. Rituximab treatment

resulted in pSS patients in a rapid decrease in peripheral B cells, accompanied by a decrease

in IgM-Rf le vels. (figure 3)

Conclusion

Salivary research provides powerful tools to diagnose diseases affecting the salivary

glands, to assess disease progression and to evaluate treatment. Important gland-specific

parameters are sialometry, sialochemistry, and histopathology. More general tools are

subjective questionnaires (e.g. VAS, MFI. SF-36) and serological parameters.

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Reference List

(1) Pijpe J, Kalk WWI, Bootsma H, Spijkervet FKL, Kallenberg CGM, Vissink A. Progression of salivary

gland dysfunction in patients with Sjögren’s syndrome. Ann Rheum Dis 2007; 66(1):107-12.

(2) van den Berg I, Pijpe J, Vissink A. Salivary gland parameters and clinical data related to the

underlying disorder in patients with persisting xerostomia. Eur J Oral Sci 2007; 115(2):97-102.

(3) Pijpe J, van Imhoff GW, Spijkervet FKL, Roodenburg JLN, Wolbink GJ, Mansour K et al. Rituximab

treatment in patients with primary Sjögren’s syndrome: An open-label phase II study. Arthritis

Rheum 2005; 52(9):2740-50.

(4) Burlage FR, Pijpe J, Coppes RP, Hemels MEW, Meertens H, Canrinus A et al. Accuracy of collecting

stimulated human parotid saliva. Eur J of Oral Sci 2005; 113(5):386-90.

(5) Martinoli C, Derchi LE, Solbiati L, Rizzatto G, Silvestri E, Giannoni M. Color Doppler sonography

of salivary glands. Am J Roentgenol 1994; 163(4):933-41.

(6) Pijpe J, Kalk WWI, van der Wal JE, Vissink A, Kluin PM, Roodenburg JLN et al. Parotid gland

biopsy compared with labial biopsy in the diagnosis of patients with primary Sjögren’s syndrome.

Rheumatology (Oxford) 2007; 46(2):335-41.

(7) Smets EM, Garssen B, Bonke B, De Haes JC. The Multidimensional Fatigue Inventory (MFI)

psychometric qualities of an instrument to assess fatigue. J Psychosom Res 1995; 39(3):315-25.

(8) Ware JE, Jr., Sherbourne CD. The MOS 36-item short-form health survey (SF-36). I. Conceptual

framework and item selection. Med Care 1992; 30(6):473-83.

(9) Fox PC, Busch KA, Baum BJ. Subjective reports of xerostomia and objective measures of salivary

gland performance. J Am Dent Assoc 1987; 115(4):581-4.

(10) Terhaard CH, Lubsen H, Rasch CR, Levendag PC, Kaanders HH, Tjho-Heslinga RE et al. The role

of radiotherapy in the treatment of malignant salivary gland tumors. Int J Radiat Oncol Biol Phys

2005; 61(1):103-11.

(11) Vissink A, Burlage FR, Spijkervet FK, Jansma J, Coppes RP. Prevention and treatment of the

consequences of head and neck radiotherapy. Crit Rev Oral Biol Med 2003; 14(3):213-25.

(12) Burlage FR, Coppes RP, Meertens H, Stokman MA, Vissink A. Parotid and submandibular/sublingual

salivary flow during high dose radiotherapy. Radiother Oncol 2001; 61(3):271-4.

(13) Meijer JM, Pijpe J, van Imhoff GW, Vissink A, Spijkervet FK, Mansour K et al. Retreatment with

rituximab in patients with active primary Sjögren’s syndrome. IXth International Symposium on

Sjogren’s Syndrome 2006.

Progression and treatment evaluation59

Shen Hu1, Jianghua Wang1, Jiska M Meijer8, Sonya Ieong1,

Yongming Xie6, Tianwei Yu1, Hui Zhou1, Sharon Henry 1,

Arjan Vissink8, Justin Pijpe8, Cees GM Kallenberg9, David

Elashoff 7, Joseph A Loo 4,5,6, David T Wong 1, 2, 3, 4, 5

Arthritis Rheum. 2007 Nov; 56(11): 3588-600

Chapter 4b

Salivary proteomic and genomic

biomarkers for primary Sjögren’s

syndrome

1School of Dentistry and Dental Research Institute, 2Division of Head & Neck Surgery/

Otolaryngology, David Geffen School of Medicine, 3Henry Samueli School of Engineering, 4Jonsson Comprehensive Cancer Center, 5Molecular Biology Institute, 6Department of

Chemistry and Biochemistry and 7School of Public Health, University of California Los

Angeles, Los Angeles, California, USA, 8 Department of Oral and Maxillofacial Surgery and 9Clinical Immunology, University Medical Center Groningen, University of Groningen, The

Netherlands

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Abstract

Objective To identify a panel of protein and messenger RNA (mRNA) biomarkers in human

whole saliva (WS) that may be used in the detection of primary Sjögren’s syndrome (pSS).

Methods Mass spectrometry and expression microarray profiling were used to identify

candidate protein and mRNA biomarkers of pSS in WS samples. Validation of the discovered

mRNA and protein biomarkers was also demonstrated using real-time quantitative

polymerase chain reaction and immunoblotting techniques.

Results Sixteen WS proteins were found to be down-regulated and 25 WS proteins

were found to be up-regulated in pSS patients compared with matched healthy control

subjects. These proteins reflected the damage of glandular cells and inflammation of the

oral cavity system in patients with pSS. In addition, 16 WS peptides (10 up-regulated and 6

downregulated in pSS) were found at significantly different levels (p< 0.05) in pSS patients

and controls. Using stringent criteria (3-fold change; p< 0.0005), 27 mRNA in saliva samples

were found to be significantly up-regulated in the pSS patients. Strikingly, 19 of 27 genes that

were found to be overexpressed were interferon-inducible or were related to lymphocyte

filtration and antigen presentation known to be involved in the pathogenesis of pSS.

Conclusion Our preliminary study has indicated that WS from patients with pSS contains

molecular signatures that reflect damaged glandular cells and an activated immune response

in this autoimmune disease. These candidate proteomic and genomic biomarkers may

improve the clinical detection of pSS once they have been further validated. We also found

that WS contains more informative proteins, peptides, and mRNA, as compared with gland-

specific saliva, that can be used in generating candidate biomarkers for the detection of pSS.

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Introduction

Sjögren’s syndrome (SS), which was first described in 1933 by the Swedish physician Henrik

Sjögren (1), is a chronic autoimmune disorder clinically characterized by a dry mouth

(xerostomia) and dry eyes (keratoconjunctivitis sicca). The disease primarily affects women,

with a ratio of 9:1 over the occurrence in men. While SS affects up to 4 million Americans,

about half of the cases are primary SS (pSS). pSS occurs alone, whereas secondary SS

presents in connection with another autoimmune disease, such as rheumatoid arthritis

or systemic lupus erythematosus (SLE). Histologically, SS is characterized by infiltration

of exocrine gland tissues by predominantly CD4 T lymphocytes. At the molecular level,

glandular epithelial cells express high levels of HLA-DR, which has led to the speculation

that these cells are presenting antigen (viral antigen or autoantigen) to the invading T cells.

Cytokine production follows, with interferon (IFN) and interleukin-2 (IL-2) being especially

important. There is also evidence of B cell activation with autoantibody production and

an increase in B cell malignancy. SS patients exhibit a 40-fold increased risk of developing

lymphoma.

SS is a complex disease that can go undiagnosed for several months to years. Although

the underlying immune-mediated glandular destruction is thought to develop slowly over

several years, a long delay from the start of symptoms to the final diagnosis has been

frequently reported. SS presumably involves the interplay of genetic and environmental

factors. To date, few of these factors are well understood. As a result, there is a lack of early

diagnostic markers, and diagnosis usually lags symptom onset by years. A new international

consensus for the diagnosis of SS requires objective signs and symptoms of dryness, including

a characteristic appearance of a biopsy sample from a minor or major salivary gland and/or

the presence of autoantibody such as anti-SSA.(2-4) However, establishing the diagnosis

of pSS has been difficult in light of its nonspecific symptoms (dry eyes and mouth) and the

lack of both sensitive and specific biomarkers, either body fluid- or tissue-based, for its

detection. It is widely believed that developing molecular biomarkers for the early diagnosis

of pSS will improve the application of systematic therapies and the setting of criteria with

which to monitor therapies and assess prognosis (e.g., lymphoma development).

Saliva is the product of 3 pairs of major salivary glands (the parotid, submandibular, and

sublingual glands) and multiple minor salivary glands that lie beneath the oral mucosa. Human

saliva contains many informative proteins that can be used for the detection of diseases.

Saliva is an attractive diagnostic fluid because testing of saliva provides several key advantages,

including low cost, noninvasiveness, and easy sample collection and processing. This biologic

fluid has been used for the survey of general health and for the diagnosis of diseases in

humans, such as human immunodeficiency virus, periodontal diseases, and autoimmune

diseases.(5-8) Our laboratory is active in the comprehensive analysis of the saliva proteome

(for more information, see www.hspp.ucla.edu), thus providing the technologies and expertise

to contrast proteomic constituents in pSS with those in control saliva.(9-11) Thus far, we have

identified over 1,000 proteins in whole saliva (WS). In addition, we have recently identified

and cataloged ~3,000 messenger RNAs (mRNA) in human WS.(12) These studies have

provided a solid foundation for the discovery of biomarkers in the saliva of patients with pSS.

We have previously demonstrated proteome- and genome-wide approaches to harnessing

saliva protein and mRNA signatures for the detection of oral cancer in humans.(13,14)

There have been continuous efforts in the search for biomarkers in human serum or

saliva for the diagnosis of pSS. Some gene products were found at elevated levels in SS

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patient sera or saliva, including β2-microglobulin (β

2m), soluble IL-2 receptor, IL-6, anti-

Ro/SSA, anti-La/SSB, and anti-α-fodrin autoantibodies.(15-20) However, none of them

individually is sensitive or specific enough to use for the confirmative diagnosis of SS.(15)

Therefore, it is crucial to use emerging proteome- and genome-wide approaches to discover

a wide spectrum of informative and discriminatory biomarkers that can be combined to

improve the sensitivity and specificity for the detection of pSS.

Patients and methods

Patient cohort

Because sample quality is critical for clinical proteomics studies, a standardized procedure,

in strict accordance with the American-European Consensus Group Criteria for SS (2),

was used for the identification and recruitment of pSS patients for this study. A diagnostic

evaluation of SS was performed in all patients and included assessments of subjective

complaints of oral and ocular dryness, sialometry (unstimulated WS), sialography,

histopathology of salivary gland tissue, serology (SSA and SSB antibodies), eye tests (rose

bengal staining and Schirmer’s test) according to the American-European classification criteria

for SS (2), and screening for extraglandular manifestations. Three of the pSS patients were

being treated with hydroxychloroquine, and 1 patient was being treated with prednisolone.

Eight patients had a focus score of >1 on examination of parotid gland biopsy tissue.

The enrolled pSS patients and healthy control subjects were well matched for age, sex,

and ethnicity. The mean ±SD age was 37.2±9.8 years in the pSS patients (n=10) and 37.0±

10.6 years in the healthy control subjects (n=10). All subjects enrolled in this study were

Caucasian women, since pSS mainly affects women. All of the enrolled control subjects

were negative for serum anti-SSA/SSB antibodies, and none of them reported any sicca

symptoms, including oral and ocular dryness.

Saliva sample collection

Samples of WS and saliva from the parotid and submandibular/sublingual glands were

collected from each pSS patient and control subject for comparative analysis. Saliva

sample collection was performed at the University Medical Center Groningen, using

our standardized saliva collection protocols. Subjects were asked to refrain from eating,

drinking, smoking, or performing oral hygiene procedures for at least 1 hour prior to the

collection. Samples were collected in the morning, at least 2 hours after eating and rinsing

the mouth with water, according to established protocols.(21,22) WS was stimulated by

chewing paraffin and was collected over a period of 15 minutes. Glandular saliva specimens

from individual parotid glands and, simultaneously, from the submandibular/sublingual glands

were collected into Lashley cups (placed over the orifices of the Stenson’s duct) and by

syringe aspiration (from the orifices of the Warton’s duct, located anteriorly in the floor of

the mouth), respectively.

After collection, the saliva samples were immediately mixed with protease inhibitors

(Sigma, St. Louis, MO) to ensure preservation of the integrity of the proteins and then

centrifuged at 2,600g for 15 minutes at 4°C. The supernatant was removed from the pellet,

immediately aliquoted, and stored at –80°C. All samples were kept on ice during the process.

Two patients who had very low submandibular/sublingual gland salivary flow rates (0.03 ml/

minute) did not produce enough submandibular/sublingual gland saliva for this study.

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Sample preparation for proteomic analysis

The saliva samples were precipitated overnight at –20°C with cold ethanol. Following

centrifugation at 14,000g for 20 minutes, the supernatants were collected and dried with

a speed vacuum for use in the peptide biomarker study. The pellet was then washed once

with cold ethanol and collected for assay of total protein using a 2-D Quant kit (Amersham,

Piscataway, NJ). We pooled saliva samples according to the total protein content from all

patients with pSS and those from all control subjects. However, both the patients and controls

were analyzed individually for the peptide profiling experiment.

Matrix-assisted laser desorption ionization–time-of- flight mass spectrometry (MALDI-TOF-MS)

Profiling of saliva peptides in 10 pSS patients and 10 matched control subjects was performed

using a MALDI-TOF-MS system (Applied Biosystems, Foster City, CA). The peptide

fraction from each patient (n=10) and control (n=10) sample was dissolved in 10 µl of 50%

acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA). The sample was mixed with α-cyano-

4-hydroxycinnamic acid (10 mg/ml in 50% ACN/0.1% TFA) at a ratio of 1:2, and 1 µl of the

mixture was spotted on the MALDI plate for measurement. Three measurements were

performed for each sample, and the signals were averaged for subsequent data analysis.

In order to achieve an accurate comparison of the MALDI-TOF-MS data between the

patient and control groups, baseline correction and Gaussian smoothing were initially

performed to eliminate broad artifacts and noise spikes. Afterward, peak alignment was

undertaken to ensure accurate alignment of the mass/charge (m/z) values across the set

of spectra, and peak normalization was performed against the total peak intensity. These

steps ensured comparability of the MALDI-TOF-MS spectra among all subjects. Subsequent

statistical analysis (t-test) was used to reveal peptides that were present at significantly

different levels in the pSS patients as compared with the control subjects.

Two-dimensional gel electrophoresis

Saliva samples from the 10 pSS patients and from the 10 control subjects were equally pooled

according to the total protein content and then precipitated using the same procedures

described above. The pellet was washed once with cold ethanol and then resuspended in

rehydration buffer. A total of 100 µg of proteins was loaded onto each gel for the 2-D gel

separation procedure. Isoelectric focusing was performed using immobilized pH gradient

strips (11 cm long, with an isoelectric point [pI] of 3-10 nonlinear) on a Protean isoelectric

focusing cell (Bio-Rad, Hercules, CA), and sodium dodecyl sulfate-polyacrylamide gel

electrophoresis was performed in 8-16% precast Criterion gels on a Criterion Dodeca Cell

(Bio-Rad). Fluorescent SYPRO Ruby stain (Invitrogen, Carlsbad, CA) was used to visualize

the protein spots.

The gel images were acquired and analyzed using PDQuest software (Bio-Rad). The

images were initially processed through transformation, filtering, automated spot detection,

normalization, and matching. The 2-D gel image was transformed to adjust the intensity of

the protein spot and filtered to remove small noise features without affecting the protein

spot. The images were then normalized based on the total density of the gel image. The 2-D

gel images of the pSS patients (master gel) and the control subjects were used as a “match

set” for automated detection of the protein spots on the gel. Within the match set, the

detected spots were reviewed manually, and the relative protein levels in the patient sample

compared with the control sample were summarized.

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Table 1 Salivary proteins differentially expressed between pSS patients and healthy control subjects, as identified by

LC-MS/MS and Mascot database searching*

Spot No. Accession Protein name Mascot

scorePeptide matched PI Mt Ratio

(pSS/ctrl)

1 IPI00295105 Carbonic anhydrase VI 163 4 6.65 35343 0.22

2 IPI00295105 Carbonic anhydrase VI 114 5 6.65 35343 0.35

3 IPI00295105 Carbonic anhydrase VI 78 2 6.65 35343 0.29

4 IPI00004573Polymeric-immunoglobulin receptor

235 5 5.58 83262 0.48

5 IPI00004573Polymeric-immunoglobulin receptor

293 7 5.58 83262 0.39

6 IPI00004573Polymeric-immunoglobulin receptor

182 4 5.58 83262 0.56

7 IPI00019038 Lysozyme C 103 2 9.38 16526 0.21

8 IPI00022974 Prolactin-inducible protein 147 3 8.26 16562 0.52

9 IPI00009650 Von Ebner’s gland protein 239 4 5.39 19238 0.32

10 IPI00032293 Cystatin C 153 3 9.0 15789 0.43

11 IPI00013382 Cystatin SN 152 3 6.82 16361 0.46

12 IPI00013382 Cystatin SN 130 3 6.82 16361 0.61

13 IPI00002851 Cystatin D 50 1 6.70 16070 0.56

14IPI00032294IPI00013382

Cystatin S Cystatin SA

166208

34

4.954.85

16 21416 445

0.65

15 IPI00007047 Calgranulin A 104 2 6.51 10828 0.53

16 IPI00007047 Calgranulin A 79 2 6.51 10828Absent in pSS

17 IPI00027462 Calgranulin B 126 4 5.71 13234 1.05

18 IPI00219806 Psoriasin 133 4 6.28 11464 1.44

19 IPI00410714Hemoglobin alpha-1 globin chain

157 5 7.96 15292Absent in control

20 IPI00218816 Hemoglobin beta chain 48 1 6.75 15988 2.73

21 IPI00218816 Hemoglobin beta chain 51 1 6.75 15988 7.58

22 IPI00007797Fatty acid-binding protein, epidermal

211 6 6.60 15155 3.21

23IPI00472762IPI00472610IPI00430840

IGHG1 proteinHypothetical proteinIg gamma-1 chain C region

333363333

141414

8.337.507.48

508225263354866

22.64

24IPI00472610IPI00550718

IGHM proteinIg gamma-1 chain C region

260257

1111

7.508.46

5327053331

Absent in control

25 IPI00465248 Alpha-enolase 409 12 6.99 47139 4.37

26 IPI00300786 Salivary alpha-amylase, frag-ment

241 5 5.73 57731 3.41

27 IPI00300786 Salivary alpha-amylase, frag-ment

230 4 5.73 57731 2.19

28 IPI00300786 Salivary alpha-amylase, frag-ment

375 7 5.73 57731 31.53

29 IPI00300786 Salivary alpha-amylase, frag-ment

260 5 5.73 57731 2.57

30 IPI00300786 Salivary alpha-amylase, frag-ment

171 5 5.73 57731 2.50

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Table 1 continued

Spot No. Accession Protein name Mascot

scorePeptide matched PI Mt Ratio

(pSS/ctrl)

31 IPI00300786 Salivary alpha-amylase, fragment 194 4 5.73 57731 11.92

32 IPI00300786 Salivary alpha-amylase, fragment 149 4 5.73 57731 1.57

33 IPI00300786 Salivary alpha-amylase, fragment 148 4 5.73 57731 4.03

34 IPI00549682 Fructose-bisphosphate aldolase A 218 4 8.75 52306 2.59

35 IP100332161 Ig gamma-1 chain C region 138 5 8.46 36083 2.54

36 IPI00215983 Carbonic anhydrase I 119 4 6.59 28852 7.4

37 IPI00218414 Carbonic anhydrase II 98 2 8.67 31337 2.11

38 IPI00013885 Caspase-14 172 5 5.44 27662 3.32

39 IPI00419424 Ig kappa chain C region 263 7 5.82 27313 1.79

40 IPI00004656 Beta-2-microglobulin 62 2 6.06 13706 2.21

41 IP100021439 Actin 461 11 5.29 41710 3.18

42 IPI00022434 Serum albumin, fragment 492 10 5.41 69321Absent in control

* Liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) analysis and Mascot database

searching were performed to identify the proteins. Shown are the theoretical isoelectric point and molecular

mass of the protein precursors, as well as the ratio of protein levels in patients with primary Sjögren’s syndrome

(SS) and matched control subjects, as detected by 2-dimensional gel electrophoresis.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) and database searching

Protein spots showing differential protein levels were excised by a spot-excision robot

(Proteome Works; Bio-Rad) and deposited into 96-well plates. Proteins in each gel spot

were reduced with dithiothreitol, alkylated with iodoacetamide, and then digested overnight

at 37°C with 10 ng of trypsin. After digestion, the peptides were extracted and stored at

-80°C prior to LC-MS/MS analysis.

LC-MS/MS analysis of peptides was performed using an LC Packings Nano-LC system

(Dionex, Sunnyvale, CA) with a nanoelectrospray interface (Protana, Odense, Denmark) and

a quadrupole time-of-flight (Q-TOF) mass spectrometer (QSTAR XL; Applied Biosystems).

A New Objective PicoTip tip (internal diameter 8 mm; New Objective, Woburn, MA) was

used for spraying, with the voltage set at 1,850V for online MS and MS/MS analyses. The

samples were first loaded onto an LC Packings PepMap C18 precolumn (300 µm x 1 mm;

particle size 5 µm) and then injected onto an LC Packings PepMap C18 column (75 µm x 150

mm; particle size 5 µm) (both from Dionex) for nano-LC separation at a flow rate of 250

nl/minute. The eluents used for LC were 1) 0.1% formic acid and 2) 95% ACN/0.1% formic

acid, and a 1%/minute gradient was used for the separation.

The acquired MS/MS data were searched against the International Protein Index (IPI)

human protein database (available at http://www.ebi.ac.uk/IPI/IPIhelp.html) using the Mascot

(Matrix Science, Boston, MA) database search engine. Positive protein identification was

based on standard Mascot criteria for statistical analysis of LC-MS/MS data.

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Immunoblotting

Western blot analysis of α-enolase was performed on the same set of saliva samples (10 pSS

and 10 control samples). Proteins were separated on 12% NuPAGE gels (Invitrogen) at 150V

and then transferred to a polyvinylidene difluoride membrane (Bio-Rad) using an Invitrogen

blot transfer cell. After saturating with 5% milk in Tris buffered saline-Tween buffer

(overnight at 4°C), the blots were sequentially incubated for 2 hours at room temperature

with polyclonal goat α-enolase primary antibody and horseradish peroxidase–conjugated

anti-goat IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The bands

were detected by enhanced chemiluminescence (Amersham) and quantified using Quantity

One software (Bio-Rad).

Profiling of salivary mRNA by high-density oligonucleotide microarray analysis

Samples of stimulated parotid gland saliva or WS from 10 pSS patients and 8 matched controls

were preserved in RNAlater reagent (Qiagen, Valencia, CA) at a 1:1 ratio and then frozen

at –80°C. Total salivary RNA was isolated from 560 µl of RNAlater-preserved saliva (280

µl of parotid gland saliva/WS and 280 µl of RNAlater) using a viral RNA mini kit (Qiagen)

as described previously (12). Isolated total RNA was treated with 2 rounds of recombinant

DNase I (Ambion, Austin, TX) digestion, and the RNA concentration was measured with a

NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The

salivary RNA quality was examined by real-time reverse transcription–polymerase chain

reaction (RT-PCR) analysis for expression of the salivary internal reference gene transcripts

S100 calcium-binding protein A8 and annexin A2 (data not shown).

For microarray study, total salivary RNA was subjected to 2 rounds of T7-based

RNA linear amplification (10). One microliter (200 ng/µl) of poly(dI-dC) (Amersham)

was added to 11 µl of the salivary RNA sample, and 2 rounds of first-strand and second-

strand complementary DNA (cDNA) synthesis were performed with a RiboAmp HS

RNA amplification kit (Arcturus, Mountain View, CA) according to the manufacturer’s

instructions. After purification, the cDNA were in vitro transcribed to RNA and then

biotinylated with GeneChip Expression 3’-Amplification Reagents for in vitro transcription

labeling (Affymetrix, Santa Clara, CA). The labeled RNA was purified with the reagents

provided with the RiboAmp HS RNA Amplification kit. The quality and quantity of amplified

RNA were determined by spectrophotometry, with optical densities at 260/280 nm > 1.9

for all samples.

Biotinylated RNA samples (15 µg each) were subsequently fragmented, and the quality

of the fragmented RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto,

CA). The Affymetrix human genome U133 Plus 2.0 array, which contains >54,000 probe

sets representing >47,000 transcripts and variants, including ~38,500 well-characterized

human genes, was applied to salivary mRNA profiling. Fragmented RNA were hybridized

overnight to the microarrays. After a high-stringency wash to remove the unbound probes,

the hybridized chips were stained and scanned according to the manufacturer’s standard

expression protocol. The scanned images were read with the Affymetrix microarray Robust

Multiarray Average (RMA) software.(23) We deposited the microarray data we obtained

into a Minimum Information About a Microarray Experiment (MIAME)–compliant database

(available at http://.mged.org/workgroups/MIAME/miame.html); the accession number is

GSE7451.

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Statistical analysis for the mRNA study

The expression microarrays were scanned, and the fluorescence intensity was measured

using Microarray Suite 5.0 software (Affymetrix). The arrays were then imported into the

statistical software R (24). After quantile normalization and RMA background correction,

the RMA expression index was computed in R using the Bioconductor routine.(25) Since

most human RNAs are not present in saliva (12), we used the present/absent call generated

by the Affymetrix Microarray Suite 5.0 software to exclude probe sets that were assigned an

“absent” call in most (>75%) of the samples. Principal components analysis was performed

to assess the information contained in the data to separate pSS and control cases. Student’s

2-tailed t-test was used for comparison of the average gene expression signal intensity between

samples from the SS patients (n=10) and controls (n=8). P values were adjusted with the

Benjamini and Hochberg false discovery rate (FDR) criterion.(26) Fold ratios between SS and

control samples were calculated for the transcripts. For the further validation study using

real-time quantitative PCR, we applied stringent criteria: an alpha level of 0.001 for the t-test,

which corresponded to a 5% FDR based on the data, and a fold ratio of 3. For functional

analysis using MAPPFinder (27), we applied an alpha level of 0.01, which corresponded to an

8% FDR, and a fold ratio of 2, to obtain a larger list of genes.

Real-time quantitative RT-PCR

The biomarker candidates generated by microarray profiling were validated by real-time

quantitative RT-PCR on the same set of samples used for the microarray analysis. All primers

used for quantitative PCR were designed with the Primer3 program and synthesized by Sigma.

Total RNA was reverse-transcribed using reverse transcriptase and gene-specific primers.

One microliter of total RNA was used in a 20-µl volume of cDNA synthesis reaction and

then subjected to the following thermal cycling conditions: 25°C for 10 minutes, 42°C for

45 minutes, and 95°C for 5 minutes. Three microliters of cDNA was used as template for

each 20-µl PCR, which contained forward primer (200 nM), reverse primer (200 nM), and

10 µl of 2 x SYBR Green PCR Master Mix (Applied Biosystems). PCRs were performed in a

96-well plate on the Bio-Rad iCycler or IQ5 instrument (95°C for 3 minutes followed by 50

cycles of 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 30 seconds). All PCRs were

performed in duplicate for all candidate mRNA.

The specificity of the PCR was confirmed according to the melting curve of each gene, and

the average threshold cycle (Ct) was examined. The relative expression of the candidate genes

was calculated according to the 2(-ΔCt) method, where ΔCt = C

t in pSS patients – C

t in controls.

The expression ratio ([pSS patients/controls] = 2(-ΔCt)) is shown as the fold change.(28)

Pathway analysis

PathwayArchitect software, version 1.1.0 (Stratagene, La Jolla, CA) was used to investigate

the functional pathways presented by the differentially expressed genes.

Results

Salivary flow rate and total salivary protein and mRNA contents in pSS patients

Patients with pSS who had been carefully diagnosed and monitored were enrolled in this

study. All 10 patients were positive for anti-SSA/Ro antibodies, and 9 of them were also

positive for anti-SSB/La antibodies. Their mean ±SD IgG level was 23.4±7.4 gm/liter, and

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their mean ±SD IgM rheumatoid factor level was 136.3±99.6 kIU/liter. These patients

exhibited significantly lower (~50%) salivary flow rates than did the age-, sex-, and ethnicity-

matched healthy control subjects. The mean ±SD stimulated salivary flow rates in the 10

pSS patients were 0.13±0.12 ml/minute for the parotid glands (per gland), 0.32±0.38 ml/

minute for the submandibular/sublingual glands, and 0.61±0.23 ml/ minute for WS. These

rates in the 10 control subjects were 0.21±0.07 ml/minute for the parotid glands (per gland),

0.78±0.36 ml/minute for the submandibular/ sublingual glands, and 1.03±0.31 ml/minute for

WS. Due to the low volume of saliva obtained from the pSS patients, the salivary proteins

were equally pooled for the 10 pSS patients and separately for the 10 control subjects for

the 2-DE analyses.

On average, the mean ±SD total protein concentrations in the controls were determined

to be 1.26±0.40 mg/ml in submandibular/sublingual gland saliva (n=8 subjects), 0.93±

0.38 mg/ml in parotid gland saliva (n=10 subjects), and 0.95±0.52 mg/ml in WS (n=10

subjects). The total protein concentrations in the pSS patients were 1.45±0.49 mg/ml in

submandibular/sublingual gland saliva (n=8 patients), 1.40±0.56 mg/ml in parotid gland saliva

(n=10 patients), and 1.38±0.37 mg/ml in WS (n=10 patients). There were consistently

higher concentrations of proteins in the SS patients (WS, submandibular/sublingual gland

saliva, and parotid gland saliva) than in the matched healthy control subjects. In addition,

saliva from the pSS patients appeared to contain a higher concentration of total RNA

than did that from the matched controls. In parotid gland saliva, the mean ±SD RNA

concentration was determined to be 5.8±3.1 µg/ml in the pSS patients and 3.±.5 µg/ml in

the controls (p=0.05). In WS, the average RNA concentration was 10.9±5.4 µg/ml for pSS

patients and 6.6±3.6 µg/ml for matched controls (p=0.057).

Discovery of candidate peptide markers for pSS

The expression of 16 WS peptides was found to be significantly different (p=0.0046–0.0441)

in pSS patients (n=10) and controls (n=10). Ten of the 16 peptides were overexpressed

(m/z 1.107, 1.224, 1.333, 1.380, 1.451, 1.471, 1.680, 1.767, 1.818, and 2.039) and 6 were

underexpressed (m/z 2.534, 2.915, 2.953, 3.311, 3.930, and 4.187) in the pSS patients. The

peptide with an m/z of 1.451 exhibited the highest up-regulation (25.9-fold) in pSS patients

(results not shown). We also compared the native peptide patterns in saliva from the parotid

and submandibular/sublingual glands between pSS patients and control subjects (results not

shown). WS was found to contain more informative peptides than did gland-specific (parotid

or submandibular/sublingual) saliva. On average, 53 MALDI peaks were observed in WS

from the 10 pSS patients, with only 24 peaks and 26 peaks detectable in saliva from their

parotid and submandibular/sublingual glands, respectively.

Findings of 2-DE of WS proteins from pSS patients and matched control subjects

Figure 1 presents the 2-DE patterns of the proteins in pooled WS samples from 10 pSS

patients and 10 control subjects. A number of proteins were found to be differentially

expressed between the patient and control groups. By performing the PDQuest analysis

and normalizing the protein spot signals, the relative levels of these proteins were

quantified. The differentially expressed proteins (figure 1, spots 1-42) were excised and

subsequently identified using in-gel tryptic digestion and LC-Q-TOF-MS. Pooled parotid

and submandibular/sublingual gland saliva from pSS patients and control subjects was also

analyzed by 2-DE (results not shown). WS was again found to be more informative than

parotid or submandibular/sublingual gland saliva for generating candidate protein biomarkers

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for the detection of pSS. A total of 325 protein spots were detected by 2-DE analysis of WS,

whereas 232 and 267 spots were detected by 2-DE analysis of parotid and submandibular/

sublingual gland saliva, respectively.

LC-Q-TOF-MS identif ication of proteins at altered expression levels

The differentially expressed WS proteins identified by LC-Q-TOF-MS and Mascot database

searching, as well as their theoretical isoelectric point (pI), relative molecular mass (Mr), IPI

accession number, the number of peptides matched, and ratios of expression levels between

the pSS patient and matched control groups are shown in table 1.

Figure 2A depicts the tandem MS spectrum of a double-charged tryptic peptide (m/z

450.3). The precursor ion was well fragmented to yield sufficient structural information

for confident identification of the peptide sequence TIAPALVSK, which originated from

α-enolase. Mascot database searching indicated that 12 peptides were matched to this

protein, resulting in a sequence coverage of 31%. Validation of α-enolase was also performed

by Western blotting of the same set of samples used for the 2-DE study. (figure 2B) An

equal amount of total proteins from each sample was used for immunoblotting of α-enolase

and actin. Both α-enolase and actin were found to be up-regulated in WS from pSS patients,

which is consistent with the 2-DE results. (table 1) P values were calculated to be 0.006 for

α-enolase without actin normalization and 0.037 with actin normalization for comparisons

between the pSS patient and healthy control groups.

Figure 1

Comparative analysis of proteins in whole saliva (WS) samples from patients with primary Sjögren’s syn-

drome (pSS) and age-, sex-, and ethnicity-matched control subjects, as determined by 2-dimensional gel

electrophoresis (2-DE) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-

TOF-MS). Shown are the 2-DE patterns of proteins in pooled WS from 10 control subjects and 10 pSS

patients. A total of 100 µg of total proteins from each pooled sample was used for the 2-D gel separation.

The differentially expressed proteins (spots 1-42; see Table 1 for the complete list) were identified using in-

gel tryptic digestion and LC-Q-TOF-MS.

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Identif ication of candidate genomic markers of pSS in saliva samples

For all the arrays, the mean ±SD percentage of genes present was 13.2±2.9%. This is similar

to the finding in our previous study (12) and indicates consistency of the techniques used

for sample preparation. Microarray profiling indicated that WS contains >10 times more

informative mRNA than does parotid gland saliva. A total of 328 mRNA had a >2-fold

change in WS from pSS patients, while only 21 mRNA had a >2-fold change in parotid

gland saliva from these patients. Therefore, we focused on the discovery and validation

of WS candidate mRNA biomarkers using microarray and real-time quantitative RT-PCR

strategies.

Gene expression profiles of individual WS samples from 10 pSS patients and 8 controls

were compared. After filtering the transcripts by the criteria of being “present” in >25% of

the samples, a total of 6,413 transcripts were retained for further analysis. This number is

consistent with our previous results, showing that only a small number of RNAs are present

in saliva (12). Principal components analysis indicated that the information contained in the

data could well segregate control subjects and pSS patients. (figure 3) We then performed

statistical testing and fold change analysis to identify differentially expressed genes. Only a

few mRNA were found at significantly lower levels in pSS patients as compared with the

controls when using a threshold of >2-fold change and a significance level of P <0.01 (FDR

0.08). Yet, by the same criteria, 162 genes showed significant up-regulation in samples from

patients with pSS.

Pathway analysis indicated that 37 genes were involved in the IFN-α pathway, and most

Table 2 Real-time quantitative RT-PCR validation of 13 genes selected from the top 27 genes found to be

differentially expressed in pSS patients and healthy control subjects*

Gene Average Ct Control

Average Ct pSS

Δ Ct (Control/pSS)

quantitative RT-PCR, fold change 2 (-ΔCt)

P value (t-test)

Microarray fold change

GIP2 44.5±1.9 35.5±2.1 9.0 495.5 <0.001 15.76

B2M 45.0±2.1 38.8±3.4 6.2 72.1 <0.001 8.67

IFIT2 41.1±2.0 35.9±2.6 5.1 35.5 <0.001 12.19

BTG2 38.5±5.3 33.5±2.0 5.0 32.4 0.01 3.22

IFIT3 43.8±0.5 39.1±2.4 4.7 25.3 <0.001 122.82

MNDA 37.3±1.2 33.7±2.1 3.7 12.7 <0.001 8.67

FCGR3B 40.6±1.5 36.9±2.2 3.6 12.5 <0.001 25.32

TXNIP 39.2±2.1 35.6±3.2 3.6 11.7 0.01 3.42

IL18 45.3±2.1 41.8±2.5 3.5 11.5 0.01 6.12

HLAB 36.4±2.7 32.9±2.0 3.5 11.2 0.01 4.34

EGR1 37.4±2.4 33.9±2.0 3.4 10.3 0.01 7.20

COP1 40.5±1.5 38.7±3.3 1.8 3.4 0.18 7.62

TNSF 39.6±0.4 38.9±2.9 0.7 1.6 0.95 8.03

* All real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were

performed in duplicate. See Patients and Methods for calculations of the fold change (primary Sjögren’s

syndrome (SS) patients/healthy controls) and threshold cycle (Ct) data.

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of them have been reported to be IFN-α or IFN-β inducible.(29;30) These results suggest

that activation of IFN pathways is involved in the pathogenesis of pSS and that the related

information is reflected in the saliva. To facilitate biomarker discovery, we narrowed the

number of candidate biomarkers by using more stringent threshold criteria of P < 0.001

(FDR 0.05) and 3-fold change. Based on these criteria, we found 27 genes that were highly

overexpressed in samples from pSS patients. These genes are sufficiently informative for

segregating the pSS patients from the control subjects. (figure 4)

Among the top 27 genes, 13 were validated by real-time quantitative RT-PCR. Eleven of

the 13 genes were found to be significantly up-regulated in pSS patients (>10-fold change),

including the IFN-inducible protein G1P2, which showed an ~500-fold change in pSS patients.

Table 2 shows the average Ct values of these genes in pSS patients and control subjects, as

well as the quantitative PCR fold change in comparison with that of microarray profiling.

Figure 2

Analysis of α-enolase by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and immunob-

lotting. A, ESI-MS/MS spectrum of the tryptic peptide TIAPALVSK (mass/charge [m/z] 450.3 atomic mass

units [amu]) from α-enolase. This protein was found to be overexpressed in whole saliva from patients with

primary Sjögren’s syndrome (pSS), as determined by 2-dimensional gel electrophoresis. B, Immunoblot-

ting of whole saliva from 10 patients with pSS and 10 age-, sex-, and ethnicity-matched control subjects for

α-enolase and actin. An equal amount of proteins from each sample was used for the immunoblots.

A

TIAPALVSK

B

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Discussion

Although saliva has been extensively explored as a source of information that can be used

in the diagnosis of pSS, most of the previously published studies mainly examined individual

components of the saliva. High-throughput profiling techniques, such as proteomics and

expression microarray analysis, enable us to explore salivary proteins and mRNA in a global

manner and may therefore provide new and deeper insights that may lead to the discovery

of salivary biomarkers for pSS. Recently, surface-enhanced laser desorption ionization time-

of-flight mass spectrometry and differential gel electrophoresis have been used to identify

very promising candidate biomarkers of SS in tears and in parotid gland saliva.(31;32) It was

found that the proteomic profile of parotid gland saliva from SS patients is a mixture of

increased inflammatory proteins and decreased acinar proteins as compared with the profile

in non-SS controls.(32)

In order to determine which oral f luid compartment is more informative for the

discovery of biomarkers that can be used to detect pSS, we used both proteomic and

microarray approaches to profile peptides, proteins, and mRNA in WS, parotid gland saliva,

and submandibular/sublingual gland saliva from each study subject. WS as a fluid includes

secretions from 3 major salivary glands, numerous minor salivary glands, and gingival fluid,

as well as cell debris. There has therefore been concern about the complex background in

WS for discovery of disease biomarkers, whereas parotid gland saliva, if collected carefully,

may contain more specific biomarkers for pSS. Yet, there are no published reports of any

advantage of using gland-specific saliva versus WS in terms of the diagnostic potential for

Figure 3

Principal components analysis of the gene expression data in patients with primary Sjögren’s syndrome (SS)

and in age-, sex-, and ethnicity-matched control subjects. Results of the principal components (PC1 and

PC2) analysis suggest that the gene expression data we obtained segregated the 8 control subjects (green

symbols) from the 10 pSS patients (black symbols).

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pSS. The findings of our study allow us to conclude that WS is more informative than

glandular saliva for generating biomarkers to be used for the detection of pSS.

Microarray profiling indicated that WS from pSS patients contained 328 mRNA with 2-fold

change in expression, whereas the parotid gland saliva from pSS patients contained only

21 mRNA with a >2-fold change in expression. Similarly, findings of the MALDI-TOF-MS

and 2-DE analyses suggested that WS from pSS patients has more informative proteomic

components than does parotid or submandibular/sublingual gland saliva. Since the salivary

flow rate varies from person to person, the peptide or protein composition among

different individuals could be affected by the very low salivary flow rate of the parotid and

submandibular/sublingual glands. With regard to the low flow rate of glandular saliva, as well

as the additional skill set and clinical time necessary to collect gland-specific saliva, WS may

be a more appropriate clinical diagnostic fluid for the discovery and detection of biomarkers

of pSS.

The panel of candidate peptide/protein markers for pSS is completely distinct from

the panel we obtained for oral cancer.(13) This suggests that the panels of discriminatory

salivary proteomic components are likely to be different for different diseases. The majority

of underexpressed proteins found in WS from pSS patients are secretory proteins, including

3 glycoforms of carbonic anhydrase VI (figure 1, spots 1-3), cystatins, lysozyme C, polymeric

immunoglobulin receptor (pIgR), calgranulin A, prolactin-inducible protein, and von Ebner

gland protein. This suggests that the level of secretory proteins in WS from pSS patients

may be directly affected by injury to salivary glandular cells. Several of these down-regulated

proteins in the WS of pSS patients, including pIgR, lysozyme C, and cystatin C, were found

up-regulated in the parotid gland saliva of pSS patients in a previously published study.

(32) This may be factual, as evidenced by our comparative analysis of parotid gland salivary

proteins in pSS patients and control subjects (results not shown). For example, in our 2-DE

study, pIgR was also found to be up-regulated in the pooled parotid gland saliva of pSS

patients as compared with the matched control subjects (results not shown). A future study

of salivary proteins from the parotid gland versus WS in the same pSS patients would be of

interest to the pSS research community.

Two glycolysis enzymes, fructose-bisphosphate aldolase A and α-enolase, were found at

elevated levels in the WS of pSS patients. Fructose-bisphosphate aldolase A plays a central

role in glucose metabolism, catalyzing either net cleavage or synthesis during glycolysis or

gluconeogenesis. Alpha-enolase is a multifunctional glycosis enzyme involved in various

processes, such as growth control, hypoxia tolerance, and allergic responses. Previously,

α-enolase was identified as an autoantigen in Hashimoto encephalopathy, which is an

autoimmune disease associated with Hashimoto thyroiditis.(33) Alpha-enolase was also

found as an autoantigen in lymphocytic hypophysitis, and serum autoantibodies directed

against α-enolase were detected in patients with lymphocytic hypophysitis as well as in

patients with other autoimmune diseases. Excessive production of autoantibodies, which are

generated as a consequence of uptake of enolase by antigen-presenting cells and subsequent

B cell activation, can potentially initiate tissue injury as a result of immune complex

deposition.(34;35) Overexpressed proteins in WS from patients with pSS also included

psoriasin, fatty acid binding protein, carbonic anhydrases I and II, salivary amylase fragments,

caspase 14, β2m, hemoglobin (β and α1 global chains), and immunoglobulins. The elevated

level of caspase 14 protein and caspases 1 and 4 RNA in pSS patients also suggested an

interesting role of apoptosis in the pathogenesis of pSS.

Our study clearly demonstrates that pSS-related gene expression signatures are present

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in saliva and they are able to differentiate pSS patients from control subjects. To the best

of our knowledge, this is the first study on the discovery of candidate salivary mRNA

markers for the detection of pSS. We identified 162 differentially expressed genes in the

saliva of pSS patients, as compared with a reported 35 and 424, respectively, identified

in 2 studies of microarray profiling of minor salivary gland biopsy tissues.(36;37) One

of the important findings of this study is that the 37 up-regulated genes in the saliva of

pSS patients were involved in the IFN pathway. This further confirmed the findings from

previous tissue- based studies and demonstrated that the IFN-inducible gene signature

associated with pSS is reflected in patients’ saliva.(36-39) Beyond the IFN-inducible genes,

the class I major histocompatibility complex is another major group of up-regulated genes

found to be common to salivary gland and WS from patients with pSS.(36,37) Other genes

reported to be of particular interest in the pathogenesis of pSS (37) that were found to be

overexpressed in saliva are proteasome subunit β type 9, guanylate binding protein 2, IFN-

induced protein 44, and IFN-inducible protein G1P2, and β2m. These common genes found

in saliva and minor salivary gland tissue from patients with pSS support our hypothesis that

saliva harbors the biomarkers for pSS.

Figure 4

Heat map of 27 mRNA that were significantly up-regulated in patients with primary Sjögren’s syndrome

(SS) as compared with the age-, sex-, and ethnicity-matched control subjects, as determined by microarray

profiling analysis. Control and SS patient numbers are shown at the bottom.

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The mechanism of IFN pathway activation in the pathogenesis of pSS may be more

complicated. Activation of IFN pathways (both type I and type II) in pSS suggests the

involvement of viral infection in its pathogenesis. Immune complexes consisting of auto-

antibodies and DNA- or RNA-containing autoantigens derived from apoptotic or necrotic

cells are also able to induce the production of type I IFN. However, IFN itself is not among

the genes we found to be overexpressed in the saliva of the pSS patients. On the other

hand, low-dose IFN-α has been reported to be effective in the treatment of some patients

with pSS. A single-blind controlled trial showed that IFN-α therapy significantly improved

salivary gland dysfunction in SS patients.(40) Serial labial salivary gland biopsy in 9 patients

responding to IFN-α therapy showed a signif icant decrease (p<0.02) in lymphocytic

infiltration and a significant increase (p=0.004) in the proportion of intact salivary gland

tissue after IFN-α treatment.(41)

Type I IFN pathway dysregulation, however, has been reported in such distinct diseases as

SLE, dermatomyositis, psoriasis, and SS (36), indicating that the consequences of activation

of this pathway are likely to be tissue type-dependent and, from a therapeutic point of view,

that local immune modulation (e.g., direct infusion into salivary glands) may be more efficient

than systemic interference. An initial viral infection-induced type I IFN production in salivary

glands, with prolonged activation triggered by autoantibodies from nucleic acid–containing

immune complexes, has been proposed as the mechanism of pSS.(42) More importantly,

activation of this IFN pathway may provide potential therapeutic targets for pSS, and saliva

may be used to monitor the response to the IFN-related target modulation.

One of the up-regulated genes seen in the saliva of patients with pSS is β2m, which is

also regulated by IFN. Significantly elevated levels of β2m have previously been detected in

saliva from patients with pSS.(43) The concentration of salivary (but not serum) β2m was

highly related to the salivary gland biopsy focus score.(43) The value of salivary β2m protein

as a biomarker for pSS has been evaluated, and it has been suggested that determination of

β2m levels in the saliva could be used as a noninvasive measurement for confirmation of the

diagnosis of SS.(44) Interestingly, but not surprisingly, we found that both the mRNA and

protein levels of β2m are concordantly overexpressed in the saliva of patients with pSS.

From the top 27 mRNA found to be overexpressed in WS from pSS patients, as revealed

bymicroarray profiling, we were able to validate 11 of the genes; expression of the other

16 genes was too low for quantitative PCR assessment. The most overexpressed mRNA

was found to be G1P2, which has a function in cell signaling and has been reported to be

up-regulated at the mRNA level in minor salivary glands from patients with pSS.(37) There

were discrepancies with regard to the fold change as determined by the quantitative PCR

and the microarray studies.

There are many factors that may contribute to the observed discrepancies, including the

procedures unique to the microarray analysis, such as nonspecific and/or cross-hybridization

of labeled targets to array probes, as well as those unique to real-time quantitative RT-

PCR, such as amplification biases.(45) Also, the increased distance between the location

of the PCR primers and the microarray probes on a given gene was found to decrease

the correlation between the 2 methods.(46) In our study, the amplified RNA used for

microarray assay and the unamplified RNA used for the real-time quantitative RT-PCR

validation studies can introduce variances in the fold change between the 2 methods.

Furthermore, we do not expect there to be perfect correlation between the fold change

as determined by quantitative PCR and by microarray analyses, since there is considerable

variability in the fold change statistic, especially in the case of genes that are near the limit

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of detection by quantitative PCR. For genes with expression levels that are too low for the

quantitative PCR techniques in current use, it is still possible that they may be validated

when the technology improves. Nevertheless, these 11 highly expressed genes, once they

are further validated in a new and independent patient cohort, may be used in the clinical

detection of pSS.

There was little correlation between the protein and mRNA markers identified. This

has been observed for biologic systems when efforts were made to correlate the gene

expression at both the protein and mRNA levels.(47;48) In a previous correlation analysis

of the human saliva proteome and transcriptome, we demonstrated that complementary

validation (e.g., Western blotting for protein or RT-PCR for mRNA) is required in

the conduct of RNA-protein correlation studies of individual genes after initial mass

spectrometry and expression microarray profiling.(49) If mutual validation is performed,

there may be higher correlations between the protein and mRNA candidate markers in

saliva identified in the present study. Nevertheless, the discrepancy we found may suggest

that the combination of both mRNA and protein markers is important for improving the

detection of pSS.

As a result of this preliminary study, a number of promising salivary protein and mRNA

candidates that are characteristic of pSS have been identified. Many of these candidate

biomarkers have not previously been associated with SS and, in combination, they may

eventually be validated as specific biomarkers of pSS, thus improving the clinical diagnosis

of pSS. Ideally, the biomarkers would be very specific for pSS and would discriminate

pSS from other autoimmune diseases of a similar immunopathologic background. Future

studies will include new pSS patients as well as patients with other autoimmune diseases

as control groups, aiming to validate the candidate genes either through the use of real-

time quantitative RT-PCR for mRNA or immunoassays for proteins. Absolute quantification

will provide a cutoff value for each biomarker selected, and combination of the mRNA and

protein markers will allow the eventual construction of a multimarker prediction model that

can be used as an adjunct to the current diagnostic criteria for the clinical diagnosis of pSS.

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(45) Chuaqui RF, Bonner RF, Best CJ, Gillespie JW, Flaig J, Hewitt SM, Phillips JL, Krizman DB, Tangrea

MA, Ahram M, Linehan WM, Knezevic V, Emmert-Buck MR. Post-analysis follow-up and validation

of microarray experiments. Nat Genet 2002;32 Suppl:509-14.

(46) Etienne W, Meyer MH, Peppers J, Meyer RA Jr Comparison of mRNA gene expression by RT-PCR

and DNA microarray. Biotechniques 2004; 36:618-26.

(47) Gygi SP, Rochon Y, Franza BR, Aebersold R. Correlation between protein and mRNA abundance in

yeast. Mol Cell Biol 1999;19:1720-30.

(48) Baliga NS, Pan M, Goo YA, Yi EC, Goodlett DR, Dimitrov K, Shannon P, Aebersold R, Ng WV, Hood

L. Coordinate regulation of energy transduction modules in Halobacterium sp. analyzed by a global

systems approach. Proc Natl Acad Sci U S A 2002;99:14913-8.

(49) Hu S, Li Y, Wang J, Xie Y, Tjon K, Wolinsky L, Loo RR, Loo JA, Wong DT. Human saliva proteome and

transcriptome. J Dent Res 2006, 85:1129-33.

Justin Pijpe1, Jiska M Meijer1, Hendrika Bootsma2, Jaqueline

E van der Wal3, Fred KL Spijkervet1, Cees GM Kallenberg2,

Arjan Vissink1, Stephan Ihrler4

Arthritis Rheum. 2009 Oct; 29;60(11):3251-6

Chapter 5b

Clinical and histologic evidence of

salivary gland restoration supports

the efficacy of rituximab treatment in

Sjögren’s syndrome

Departments of 1Oral and Maxillofacial Surgery, 2Rheumatology and Clinical Immunology, and 3Pathology, University Medical Center Groningen, University of Groningen, The Netherlands

and 4Ludwig Maximilian university, Institute of pathology, München, Germany

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Abstract

Objective To assess the effect of rituximab (anti-CD20 antibody) therapy on the (immuno)

histopathology of parotid tissue in patients with primary Sjögren’s syndrome (pSS) and the

correlation of histologic findings with the flow rate and composition of parotid saliva.

Methods In a phase II study, an incisional parotid biopsy specimen was obtained from 5

patients with pSS before and 12 weeks after rituximab treatment (4 infusions of 375 mg/

m2). The relative amount of parotid parenchyma, lymphocytic infiltrate and fat, and the

presence/quantity of germinal centers and lymphoepithelial duct lesions were evaluated.

Immunohistochemical characterization was performed to analyze B:T cell ratio of the

lymphocytic inf iltrate (CD20, CD79a, CD3) and cellular proliferation in the acinar

parenchyma (by double immunohistologic labeling for cytokeratin 14 and Ki-67). Histologic

data were correlated to parotid flow rate and saliva composition.

Results Four patients showed an increased salivary flow rate and normalization of the initially

increased salivary sodium concentration. Following rituximab treatment, the lymphocytic

infiltrate was reduced, with a decreased B:T cell ratio and (partial) disappearance of

germinal centers. The amount and extent of lymphoepithelial duct lesions decreased in

3 patients and was completely absent in 2 patients. The initially increased proliferation of

acinar parenchyma in response to the inflammation was reduced in all patients.

Conclusion Sequential parotid biopsy specimens obtained from patients with pSS before and

after rituximab treatment demonstrated histopathologic evidence of reduced glandular

inflammation and redifferentiation of lymphoepithelial duct lesions to regular striated ducts

as a putative morphologic correlate of increased parotid flow and normalization of salivary

sodium content. These histopathologic findings in few patients underline the efficacy of B

cell depletion and indicate the potential for glandular restoration in SS.

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Introduction

Currently, there is no evidence-based intervention treatment for Sjögren’s syndrome (SS),

but biologic agents are promising.(1) Rituximab, a chimeric murine/human anti-CD20

monoclonal antibody that binds to the B-cell surface antigen CD20, is a well-established

therapeutic agent in the treatment of B-cell non-Hodgkin lymphomas, and is a new

promising therapeutic modality in different autoimmune disorders, such as rheumatoid

arthritis (RA) and systemic lupus erythematosus.(2)

The salivary glands of patients with SS are histologically characterized by lymphocytic

infiltration with progressive parenchymal atrophy and formation of the characteristic

lymphoepithelial lesions in striated ducts, formerly called “epimyoepithelial lesions”.(3) Our

group has previously shown that lymphoepithelial lesions develop from basal cells of striated

ducts, representing an aberrant metaplastic differentiation, triggered by the epitheliotropic

autoimmune inflammation in SS.(3) In parallel, parenchymal acinar cells in SS demonstrate

increased proliferation in an effort to partially compensate for enhanced apoptotic cell

loss.(3-5) Our group previously reported clinical data from a phase II trial with rituximab

treatment in 8 patients with primary SS, which showed significant improvement of subjective

symptoms and increased salivary secretion with partial normalization of increased sodium

concentration of saliva in patients with early-onset SS.(6) These findings might indicate

partial recovery of salivary gland tissue.(7) In 5 of the 8 patients with pSS involved in the

above-mentioned study, sequential parotid gland biopsy specimens were available for

histologic analysis; these specimens were obtained before and 12 weeks after rituximab

treatment. In the other 3 patients with pSS no second biopsy specimen was obtained,

because these patients did not complete rituximab treatment due to the development of

serum sickness.(6)

The biopsy material gave us the unique opportunity to correlate clinical f indings,

including the salivary flow rate and composition of saliva, with the findings of a detailed

immunohistopathologic analysis of the parotid gland biopsy specimens obtained before and

after rituximab treatment in order to histologically verify the effects of therapeutic B cell

depletion in patients with SS.

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Patients and methods

Study design

Five female patients (mean age 53 years, range 43-65 years), all of whom fulfilled the

American-European consensus criteria for pSS, were treated with 4 infusions rituximab

(Roche, Woerden, the Netherlands), given at a dosage of 375 mg/m2/week. No other

immunosuppressive therapy was used. An incisional biopsy specimen of the parotid gland

was obtained from the same gland before and 12 weeks after therapy.(8) These patients

were part of an earlier reported phase II trial.(6)

Parotid gland function and salivary composition

Unstimulated and stimulated parotid saliva was collected in a standardized way at baseline

and 12 weeks after treatment as described previously.(6) Flow rates were calculated and

sialochemical analysis was performed, focusing on the concentration of sodium in parotid

saliva, particularly because increased sodium in parotid saliva is indicative of SS and reflects

damage to the ductal system. High levels of sodium in the saliva of patients with SS are

associated with higher levels of disease activity and a more progressive course of the

disease.(9)

Histopathologic analysis

Biopsy specimens were fixed in 4% neutral buffered formalin, embedded in paraffin, cut at a

thickness of 3 µm, and stained with hematoxylin and eosin. The relative amount of glandular

parenchyma, lymphocytic infiltrate, and fat was assessed semiquantitatively in steps of 10%,

each in relation to the total amount of biopsied parotid tissue. The presence of secondary

germinal centers was assessed as follows: 0 = no germinal centers, I = few (mostly small)

germinal centers, and II = many (often large) germinal centers. The characteristic ductal

alterations of SS (lymphoepithelial lesions) were evaluated by the following grading system: 0

= none, I = few and partially developed lymphoepithelial lesions (not circumferential, <50%

of all striated ducts) and II = fully developed lymphoepithelial lesions (fully circumferential,

>50% of all striated ducts). Biopsy specimens were independently scored as based on these

criteria by 2 investigators (J.P. and S.I.) in a blinded manner. In case of discrepancy a definite

score was determined by consensus.

Immunohistochemical analysis

In representative areas of lymphocytic infiltrates the numbers of B cells (staining for CD20)

and T cells (CD3) were quantified in 1000 lymphocytes each, and consecutively calculated

as B/T cell ratio. To evaluate a possible additional down-regulation of CD20 antigen

presentation on persisting B cells due to anti-CD20 therapy, quantification of B cells was

separately performed with antibodies to CD20 and CD79a. Due to technical limitations, it

is not possible to quantify the absolute amount of B and T cells.

As described previously (4), a double immunohistochemical labeling technique for

cytokeratin 14 (CK14) (labeling basal cells of striated ducts and myoepithelial cells)

and Ki67 (labeling cellular proliferation) greatly enhances the exact identification and

quantification of cellular proliferation of the various epithelial cells of the gland. In order

to evaluate the regenerative potential of the glandular parenchyma, cellular proliferation

in the CK14-negative acinar cells was calculated in representative areas of lymphocytic

infiltration (nuclear positivity for Ki67 as a percentage of 400 acinar cells). For staining of

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Figure 1

Comparison of parotid biopsy specimens obtained from patient 3 before therapy (left, f igures 1A1-4) and

12 weeks after therapy (right, f igure 1B1-4) Magnif ication 120x figures 1A1,1B1; 100x figures 1A2,1B2; 60x

f igures 1A3,1B3; 200x f igures 1A4,1B4. Figure 1A1: Before treatment, double staining illustrates intense

inflammation (arrows) with highly proliferating, large germinal centres (GS; Ki67 with nuclear staining), fully

developed lymphoepithelial lesions (LEL; CK14 staining) and reduced glandular parenchyma (PAR). After

therapy (f igure 1B1), inf lammation is reduced (arrows) with absence of germinal centres and presence of

regular striated ducts (SD) devoid of lymphoepithelial lesions. Before therapy there was a dominance of B

lymphocytes with germinal centres (GS; f igure 1A2: CD20) in comparison to T lymphocytes (inset: f igure

1A3: CD3). After therapy the overall reduced lymphoid inf iltrate with slight dominance of T lymphocytes

(inset: f igure 1B3: CD3) in comparison to B lymphocytes (f igure 1B2: CD20). In higher magnif ication

(f igure 1A4) fully developed lymphoepithelial lesions, many intraepithelial lymphocytes and increased

basal cell proliferation (arrows), contrasting after therapy to regular striated duct with CK14-positive basal

cells (arrows in f igure 1B4) with regular differentiation into luminal ductal cells, devoid of intraepithelial

lymphocytes (arrowheads).

Chapter 5b

88

Table 1 Clinical and (immuno-)histological data before and after rituximab therapy.

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Tendency

Before After Before After Before After Before After Before After

Clinical findings

Parotid flow 0.14 0.16 0.15 0.21 0.18 0.22 0.17 0.20 0.01 0.02 ↑

Na+ in parotid saliva 39 (5) 27 (2) 12 (5) 7 (7) 19 (6) 8 (7) 4 (6) 3 (7) N.A. N.A. ↓

Histopathology

Parenchyma (%)* 20-30 10-20 70-80 40-50 50-60 40-50 60-70 60-70 70-80 70-80 ↓

Lymphocytic infiltrate (%)* 60-70 40-50 10-20 10-20 20-30 0-10 20-30 0-10 10-20 10-20 ↓

Fat (%)* 10-20 20-30 10-20 50-60 20-30 40-50 20-30 20-30 0-10 0-10 ↑

Germinal centres II II I No II No I No No No ↓

Lymphoepithelial duct lesions (LEL)

II I I I II No I I I No ↓

Proliferation of acinar parenchyma in % (Ki67)

3.8 3.2 3.4 2.3 3.5 2.5 1.8 1.2 4.7 3.5 ↓

B :T cell ratio (CD20/CD3) 76/24 67/33 59/41 28/72 58/42 35/65 54/45 52/48 43/57 35/65 ↓

Parotid f low: stimulated parotid secretion (ml/min); Na: concentration of sodium in parotid saliva (mmol/l), value in brackets: sodium concentration to be ex-

pected in healthy subjects with the given parotid f low; N.A.: not available; * Percentages in steps of 10% represent assessment of the area of the biopsy specimen.

Germinal centres, I = few germinal centres, II = many germinal centres; Lymphoepithelial duct lesions, I = partially developed lymphoepithelial lesions (not cir-

cumferential, <50% of all ducts), II = fully developed lymphoepithelial lesions (fully circumferential, >50% of all ducts); B:T cell ratio: ratio of B and T lymphocytes

in the infiltrate; ↑: increase, ↓: decrease.

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CK14 an aividin-biotin-peroxidase method was applied (ABC kit; Vector, Burlingame, CA),

for staining of Ki67, the alkaline phosphatase-anti-alkaline phosphatase method was used

(APAAP-ChemMate; Dako Cambridge, UK).

Results

The clinical and (immuno)histologic data for biopsy specimens obtained before and after

rituximab treatment are summarized in table 1. All patients showed a clinical response as

reflected, among other factors, by significant improvement of subjective symptoms.(6) Four

of 5 patients showed a minor-to-moderate increase in the parotid flow rate (mean increase

24%). The baseline sodium concentration in parotid saliva was increased in the saliva

samples from these 4 patients (patient 5 had no salivary parotid flow at baseline) (table

1). The sodium concentration decreased after treatment in all 4 of the above-mentioned

patients, and values returned to near normal in 2 of these 4 patients.

The histologic data showed a tendency towards reduced lymphocytic infiltration after

therapy with a decrease of the B:T cell ratio, indicating a major decrease especially in the

number of B lymphocytes, in combination with a reduction of germinal centers (which were

completely absent in 4 patients), (figures 1A2, 1B2, 1A3, 1B3). The number of B lymphocytes

based on staining for CD20 and CD79a did not differ. The amount of acinar parenchyma

did not change or was slightly decreased, and the amount of fat did not change or was

increased. Parallel to the reduction in the number of intraepithelial lymphocytes, the amount

and extent of lymphoepithelial lesions decreased in 3 of 5 patients, and these lesions were

completely absent in 2 of 5 cases (figures 1A1, 1B1, 1A4, 1B4). Cellular proliferation of acinar

parenchyma before therapy was higher (average 3.4% figure 2A) than that of normal acinar

parenchyma of patients without SS (2.0%,(5)), and was found to be reduced in all patients

after therapy (on average 2.5% ). The most significant improvement of clinical and histological

findings was observed in patient 3, (as shown in figures 1 and 2A). Statistical correlation of the

different parameters could not be determined due to the small sample size.

Discussion

Rituximab is a promising treatment option for patients with pSS and systemic complications

and/or active and progressive disease, but more data from randomized controlled trials are

warranted before more accurate conclusions on the role of rituximab can be made.(10)

This study is the first to present histologic data demonstrating evidence of a reduction

in glandular inflammation combined with signs of partial glandular restoration, parallel

to increased parotid saliva flow and normalization of initially increased levels of salivary

sodium. As expected, the reduction of inflammation was mainly attributable to a depletion

of B lymphocytes, as has been previously described following rituximab therapy in

RA.(11) Although quantification of the absolute amount of B and T cells was not possible

for technical reasons, the overall decrease in the amount of infiltrate, combined with

a decreased B:T cell ratio, suggests a relevant decrease in the amount of B cells. The

preponderant absence of germinal centers and the reduction of intraepithelial lymphocytes

in the salivary ducts after therapy underline the significant reduction of inflammatory

activity. This correlates to complete depletion of B cells in the peripheral blood 12 weeks

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after start of treatment (6), comparable to data from a recent study in patients with

RA.(12) In addition, also T lymphocytes seemed to decrease slightly after therapy, although

this could not be quantified.

Although the number of B cells in parotid gland tissue was decreased, B cells were not

completely depleted. The discrepancy with complete B cell depletion observed in peripheral

blood might be explained by the expression of different protective factors in this tissue,

such as BLyS (B lymphocyte stimulator) or BAFF (B cell activating factor). The same

phenomenon has been observed in patients with RA treated with rituximab.(12) In contrast,

another study of SS patients showed a complete depletion of B cells in labial salivary glands 4

months after rituximab treatment.(13) Possible explanations for this difference might be the

increased inflammatory activity in parotid salivary glands (reflected by germinal centers) or

a difference in the expression of BAFF or BLyS. Further studies are necessary to investigate

the different B cell subsets before and after treatment and the expression of BAFF or BLyS.

The widespread presence of fully developed lymphoepithelial lesions before therapy

and the reduction or complete disappearance of lymphoepithelial lesions after therapy

offer histopathologic evidence that fully developed lymphoepithelial lesions can completely

redifferentiate into regular striated ducts (see figure 2B). As shown previously by our

group lymphoepithelial lesions in SS develop from enhanced proliferation of basal cells of

striated ducts with an aberrant metaplastic lymphoepithelial differentiation, triggered by

the epitheliotropic autoimmune inflammation.(3;14) Supposedly, this redifferentiation into

regular striated ducts after therapy is recruited from surviving and proliferating basal cells

in lymphoepithelial lesions with physiological differentiation into regular ductal cells (figure

1B4).

Figure 2

A. Minor increase of acinar cell proliferation (arrows) as demonstrated by double staining with CK14-Ki67,

adjacent to lymphocytic infiltration with lymphoepithelial lesions (LELs) and germinal centre (GS; patient 3

prior to therapy; magnification 200x).

B. Schematic illustration of partially reversible glandular alterations in SS: Black arrows (bottom) indicate

transformation of striated duct (left) into incomplete (middle) and fully developed lymphoepithelial lesions

(right), in addition to progressive loss of acini and intercalated ducts (black arrows top). Grey arrows

(bottom) illustrate evidence of complete redifferentiation of fully developed lymphoepithelial lesions to

regular striated ducts after therapy. Effective regeneration of intercalated ducts and acini as an effect of

successful Rituximab therapy is hypothetical (dotted grey arrows).

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In healthy subjects, most of the high sodium content in primary saliva is actively

reabsorbed during passage through striated ducts. The increased sodium content in saliva of

patients with pSS has been attributed to severely impaired reabsorption in the structurally

altered lymphoepithelial lesions.(9) Reduction or normalization, respectively, of the salivary

sodium concentration after B cell depletion obviously is attributable to partial or complete

redifferentiation of lymphoepithelial lesions to regular striated ducts, with reconstituted

physiological function, including regular reabsorption of sodium.

Increased proliferation of acinar parenchyma in pSS in comparison with regular glands has

been interpreted as a regenerative effort to compensate for increased apoptotic cell loss in

the inflamed parenchyma.(5) Accordingly, the observed minor decrease of proliferation in

acinar parenchyma after rituximab treatment of pSS presumably is attributable to a decrease

of the inflammatory stimulus. There is no good explanation for the almost absent parotid

salivary flow in patient 5, despite the amount of salivary parenchyma (table 1). It has been

shown that many patients with SS have, within their salivary glands, large amounts of acinar

tissue that is unable to function in vivo, possibly due to antimuscarinic antibodies.(9;15)

In summary, these f indings are the f irst to provide histopathologic evidence that

rituximab treatment in SS can induce reduction of glandular inflammation and structural

redifferentiation of lymphoepithelial duct lesions, correlating to a gain in function of the

glands, especially with respect to improved function of the structurally redifferentiated

striated ducts. The decrease in lymphocytic infiltration, the number of germinal centers,

intraepithelial lymphocytes, and acinar proliferation, combined with redifferentation of

lymphoepithelial lesions in 3 patients, suggests efficacy of B cell depletion in salivary glands.

A larger placebo-controlled randomized clinical trial investigating the immunohistologic

correlation of sequential biopsy specimens obtained before and after therapy has been

started by our group in order to prove the findings suggested in this uncontrolled study.

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Reference List

(1) Meijer JM, Pijpe J, Bootsma H, Vissink A, Kallenberg CGM. The future of biologic agents in the

treatment of Sjögren’s syndrome. Clin Rev Allergy Immunol 2007; 32:292-297.

(2) Edwards JC, Cambridge G. B-cell targeting in rheumatoid arthritis and other autoimmune diseases.

Nat Rev Immunol 2006; 6:394-403.

(3) Ihrler S, Zietz C, Sendelhofert A, Riederer A, Lohrs U. Lymphoepithelial duct lesions in Sjögren-type

sialadenitis. Virchows Arch 1999; 434:315-323.

(4) Ihrler S, Zietz C, Sendelhofert A, Lang S, Blasenbreu-Vogt S, Lohrs U. A morphogenetic concept of

salivary duct regeneration and metaplasia. Virchows Arch 2002; 440:519-526.

(5) Ihrler S, Blasenbreu-Vogt S, Sendelhofert A, Rossle M, Harrison JD, Lohrs U. Regeneration in chronic

sialadenitis: an analysis of proliferation and apoptosis based on double immunohistochemical

labelling. Virchows Arch 2004; 444:356-361.

(6) Pijpe J, Van Imhoff GW, Spijkervet FKL, Roodenburg JLN, Wolbink GJ, Mansour K et al. Rituximab

treatment in patients with primary Sjögren’s syndrome: An open-label phase II study. Arthritis

Rheum 2005; 52:2740-2750.

(7) Pijpe J, Van Imhoff GW, Vissink A, Van der Wal JE, Kluin PM, Spijkervet FKL et al. Changes in

salivary gland immunohistology and function after rituximab mono-therapy in a patient with

Sjögren’s syndrome and associated MALT-lymphoma. Ann Rheum Dis 2005; 64:958-960.

(8) Pijpe J, Kalk WWI, Van der Wal JE, Vissink A, Kluin PM, Roodenburg JLN et al. Parotid gland

biopsy compared with labial biopsy in the diagnosis of patients with primary Sjögren’s syndrome.

Rheumatology (Oxford) 2007; 46:335-341.

(9) Baum BJ. Principles of saliva secretion. Ann N Y Acad Sci 1993; 694:17-23.

(10) Isaksen K, Jonsson R, Omdal R. Anti-CD20 treatment in primary Sjögren’s syndrome. Scand J

Immunol 2008; 68:554-564.

(11) Vos K, Thurlings RM, Wijbrandts CA, van SD, Gerlag DM, Tak PP. Early effects of rituximab on the

synovial cell infiltrate in patients with rheumatoid arthritis. Arthritis Rheum 2007; 56:772-778.

(12) Thurlings RM, Vos K, Wijbrandts CA, Zwinderman AH, Gerlag DM, Tak PP. Synovial tissue response

to rituximab: mechanism of action and identification of biomarkers of response. Ann Rheum Dis

2008; 67:917-925.

(13) Pers JO, Devauchelle V, Daridon C, Bendaoud B, Le BR, Bordron A et al. BAFF-modulated

repopulation of B lymphocytes in the blood and salivary glands of rituximab-treated patients with

Sjögren’s syndrome. Arthritis Rheum 2007; 56:1464-1477.

(14) Palmer RM, Eveson JW, Gusterson BA. `Epimyoepithelial’ islands in lymphoepithelial lesions. An

immunocytochemical study. Virchows Arch A Pathol Anat Histopathol 1986; 408:603-609.

(15) Dawson L, Tobin A, Smith P, Gordon T. Antimuscarinic antibodies in Sjögren’s syndrome: where

are we, and where are we going? Arthritis Rheum 2005; 52:2984-2995.

Salivary gland restoration93

Jiska M Meijer1, Justin Pijpe1, Arjan Vissink1, Cees GM

Kallenberg2, Hendrika Bootsma2

Ann Rheum Dis. 2009 Feb;68(2):284-5

Chapter 5a

Treatment of primary Sjögren’s syndrome

with rituximab: extended follow-up,

safety and efficacy of retreatment

Departments of 1Oral and Maxillofacial Surgery, 2Rheumatology and Clinical Immunology,

University Medical Center Groningen, University of Groningen, the Netherlands

Chapter 5a

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Introduction

We previously reported that B cell depletion therapy with rituximab (4 weekly infusions of

375 mg/m2, premedication: 25 mg prednisolone intravenously) in eight patients with early

primary Sjögren’s syndrome (pSS) and 7 patients with mucosa-associated lymphatic tissue

(MALT)/pSS was effective in reducing subjective and objective symptoms after 12 weeks of

follow-up.(1) Three patients with early pSS developed serum sickness-like disease, of whom

one patient declined to further participate. The MALT component of six of the 7 patients

with MALT/pSS was initially effectively treated with rituximab, one of these six patients

was successfully retreated 9 months after the first treatment and all six patients are still in

remission of MALT > 2 years after treatment.

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Patients and methods

We focused the present work on the extended follow-up and retreatment of the patients

with early pSS. For seven of the eight patients with early pSS, 48 week follow-up data

were available. In addition, f ive patients, who did not develop serum-sickness and in

whom symptoms returned, were retreated with four infusions of rituximab and followed

for another 48 weeks. Return of symptoms included decrease of salivary flow, increase of

rheumatoid factor and return of B cells and subjective symptoms.

Figure 1

Increase and decrease (mean values of 5 patients with primary Sjögren syndrome (pSS)) in stimulated sub-

mandibular/sublingual flow rate, IgM-rheumatoid factor (RF), B cells, visual analogue scale (VAS) score for

dry mouth during the night and Multidimensional Fatigue Inventory (MFI) score for fatigue following rituxi-

mab (re)treatment (baseline is 100%). Mean (SD) baseline values (week 0 first treatment) were: stimulated

submandibular/sublingual flow rate 0.09 (0.07) ml/min, IgM-RF 339 (329) klU/l, B cells 0.19 (0.09) 109/liter,

VAS score for dry mouth during the night 85 (12), MFI score for fatigue 16 (3).

   ● Stimulated submandibu-

lar/sublingual salivary

flow rate

    ♦ IgM-RF

∗ B cells

▲ VAS score for dry mouth

during the night

▼ MFI score for fatigue

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Results

First course of rituximab (n=7)

Depletion of peripheral B cells was complete 5 weeks after onset of therapy. By 36 weeks,

median peripheral B cell numbers had returned, although levels were still low in some

patients. Stimulated submandibular/sublingual salivary flow showed a significant increase

at week 12, followed by a gradual decline to just above baseline at 48 weeks. Similarly, a

significant improvement of most of the visual analogue scale (VAS) scores for dry mouth and

most domains of the Multidimensional Fatigue Inventory (MFI) was observed, followed by a

gradual decline to near baseline.

Retreatment with rituximab (n=5, figure 1)

Retreatment had a significant effect on B cells, levels of IgM-rheumatoid factor (RF) and

stimulated submandibular/sublingual salivary flow similar to the effects of the first course.

VAS scores for dry mouth, MFI scores for general fatigue and SF-36 questionnaire scores

for physical functioning improved significantly too. For the other subjective symptoms a

similar trend towards improvement was seen as after the first course. Again, almost all

variables had returned to baseline 6-9 months after retreatment. One patient developed

serum sickness-like disease (purpura, arthralgia, myalgia) after the second rituximab infusion

during the retreatment course. Rituximab treatment was stopped, pain relief (non-steroidal

anti-inflammatory drugs (NSAIDs)) and 120 mg methylprednisolone was given once. The

patient recovered completely.

Discussion

Rituximab appeared to be effective for at least 6-9 months in patients with pSS with active

disease, improving both subjective and objective symptoms. Development of serum sickness-

like disorder in a substantial number of patients with pSS indicates that higher doses of

corticosteroids might be needed during treatment. Retreatment resulted in a good clinical

response in patients with pSS comparable to the response in patients with RA (2) and

patients with systemic lupus erythematosus (SLE).(3) Based on these promising results, one

might consider maintenance treatment. The best approach to and timing of maintenance

treatment has, however, to be studied in future trials. Furthermore, attention has to be paid

to among others the possibility of development of humoral immunodeficiency related to

repeated treatment.(2)

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Reference List

(1) Pijpe J, van Imhoff GW, Spijkervet FKL, Roodenburg JLN, Wolbink GJ, Mansour K et al. Rituximab

treatment in patients with primary Sjögren’s syndrome: An open-label phase II study. Arthritis

Rheum 2005; 52(9):2740-50.

(2) Popa C, Leandro MJ, Cambridge G, Edwards JC. Repeated B lymphocyte depletion with rituximab

in rheumatoid arthritis over 7 yrs. Rheumatology (Oxford) 2007; 46(4):626-30.

(3) Smith KG, Jones RB, Burns SM, Jayne DR. Long-term comparison of rituximab treatment for

refractory systemic lupus erythematosus and vasculitis: Remission, relapse, and re-treatment.

Arthritis Rheum 2006; 54(9):2970-82.

Rituximab retreatment10

1

Jiska M Meijer1, Petra Meiners1, Arjan Vissink1, Fred KL

Spijkervet1, Wayel Abdulahad2, Nicole Kamminga3, Liesbeth

Brouwer2, Cees GM Kallenberg2, Hendrika Bootsma2

Arthritis Rheum. 2010 Jan 13. (Epub ahead of print)

Departments of 1Oral and Maxillofacial Surgery, 2Rheumatology and Clinical Immunology,

and 3Opthalmology, University Medical Center Groningen, University of Groningen, The

Netherlands

Chapter 5c

Effectiveness of rituximab

treatment in primary Sjögren’s

syndrome: a randomized, double-

blind, placebo-controlled trial

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Abstract

Objective To study the efficacy and safety of B cell depletion with rituximab, a chimeric

murine/human anti-CD20 monoclonal antibody, in a double-blind, randomized, placebo-

controlled trial of patients with primary Sjögren’s syndrome (pSS).

Methods Patients with active pSS, as determined by the revised European-US criteria, and a

stimulated whole saliva secretion ≥0.15 ml/min, were treated with either rituximab (1000

mg) or placebo infusions at days 1 and 15. Patients were assigned randomly in a 2:1 ratio

(rituximab:placebo). Follow-up was conducted at 5, 12, 24, 36 and 48 weeks. The primary

endpoint was stimulated whole salivary flow rate; secondary endpoints included functional,

laboratory and subjective variables.

Results Thirty patients (29 female) were randomly allocated to treatment. Mean ages in the

rituximab and placebo groups were 43±11 and 43±17 years, and disease duration was 63±50

and 67±63 months, respectively. In the rituximab group, significant improvements, in terms

of the mean change from baseline compared with that in the placebo group were found for

the primary endpoint of secretion of stimulated whole saliva (p=0.038), and for various

laboratory parameters (B cells, rheumatoid factor), subjective parameters (multidimensional

fatigue inventory (MFI) scores and visual analogue scale (VAS) scores for sicca complaints)

and extraglandular manifestations. Moreover, rituximab treatment significantly improved

stimulated whole saliva secretion (p=0.004) and several variables (e.g., B cells, rheumatoid

factor, unstimulated and stimulated whole saliva, lissamine green test, MFI, short-form 36

(SF-36) and VAS scores), compared with baseline values. One patient developed mild serum

sickness-like disease.

Conclusions This study indicated that rituximab is an effective and safe treatment modality

for patients with pSS.

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Introduction

Sjögren’s syndrome (SS) is a systemic auto-immune disease characterized by chronic inflam-

mation of the salivary and lachrymal glands, resulting in xerostomia and keratoconjunctivitis

sicca in about 95% of patients.(1) These symptoms are frequently accompanied by

extraglandular manifestations (EGM) such as Raynaud’s phenomenon, arthritis, arthralgia

and myalgia , and 85% of the patients suf fer from severe fatigue. Moreover, B cell

hyperactivity, reflected by increased serum levels of IgG and IgM-rheumatoid factor (IgM-

RF) and the presence of anti-SS-A and anti-SS-B autoantibodies, is a common finding in

SS. Furthermore, SS has a large impact on health-related quality of life, employment and

disability as reflected by lower SF-36 scores and employment rates, and higher disability

rates in SS patients relative to the general population.(1)

To date, no causal systemic treatment has been available for SS. In pilot trials, however, it

has been shown that rituximab, a chimeric murine/human anti-CD20 monoclonal antibody

which binds to the B cell surface antigen CD20, might improve subjective and objective

symptoms related to primary SS (pSS) for at least 6-9 months.(2;3) Based on these

promising results, a randomized, double-blind, placebo-controlled trial was performed to

investigate the efficacy and safety of rituximab in the treatment of patients with pSS.

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Patients and methods

Study design

This was a prospective, single-centre, randomized, double-blind, placebo-controlled study.

The study protocol was approved by the institutional review board of the University Medical

Center Groningen. All patients provided written informed consent.

Patients

All patients were ≥18 years and fulfilled the European–US criteria for pSS.(4) Eligibility

criteria were a stimulated whole saliva secretion ≥0.15 ml /min and positivity for

autoantibodies ( IgM-RF ≥10 klU/l and anti-SS-A and /or anti-SS-B autoantibodies) .

A recent salivary gland biopsy (≤12 months before inclusion) showing characteristic

features of SS must be available.(5) During the study, patients were asked to use reliable

methods of contraception. Secondary SS patients and pSS patients who had been treated

previously with other monoclonal antibodies were excluded. Treatment with prednisone

and hydroxychloroquine had to be discontinued at least one month before baseline, and

treatment with methotrexate, cyclophosphamide, cyclosporin, azathioprine and other

disease-modifying anti-rheumatic drugs at least 6 months before baseline. Patients were

allowed to use artificial tears and artificial saliva, but the regimen had to remain identical

during follow-up. The use of these substitutes had to be stopped one day prior to each

assessment. All patients underwent a baseline electrocardiogram and chest radiography.

Patients with a history of any malignancy, with underlying cardiac, pulmonary, metabolic,

renal or gastrointestinal conditions, with chronic or latent infectious diseases, or with

immune deficiency were excluded.

Drug administration

Twenty patients were treated with intravenous (i.v.) infusions of 1000 mg rituximab (Roche,

Woerden, The Netherlands) and 10 patients were treated with i.v. placebo infusions on days

1 and 15. To minimize side effects (infusion reactions, serum sickness), all patients were

pre-medicated with methylprednisolone (100 mg/i.v.), acetaminophen (1000 mg/p.o.) and

clemastine (2 mg/i.v.), and received 60 mg oral prednisone on days 1 and 2, 30 mg on days 3

and 4, and 15 mg on day 5 after each infusion.

Outcome parameters

The primary endpoint was defined as a significant improvement of secretion of stimulated

whole saliva (ml/min) in the rituximab group compared with the placebo group.

Secondary endpoints were salivary/lachrymal function, and immunological and subjective

variables. All variables were assessed at baseline (within 4 weeks before treatment), and at

weeks 5, 12, 24 and 48 after treatment.

Salivary gland function

Whole, parotid and submandibular/sublingual saliva were collected in a standardized manner

and at a fixed time of the day (in this study between 1 and 4 p.m.) in order to minimize

fluctuations related to a circadian rhythm of salivary secretion (6;7) and composition.

Glandular saliva was collected from both individual parotid glands by use of Lashley cups

and submandibular/sublingual saliva was collected simultaneously by syringe aspiration from

the area with the orofices of the submandibular excretory ducts. Unstimulated saliva was

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collected the first 5 minutes, followed by stimulated saliva for 10 minutes. The salivary glands

were stimulated by citric acid solution (2%), applied with a cotton swab to the lateral borders

of the tongue every 30 seconds. Flow rates were calculated and composition of saliva was

analyzed according to the methods described by Burlage et al. and Kalk et al.(8-10)

Lachrymal gland function

Lachrymal gland function was evaluated by performing a Schirmer’s test, a lissamine green

(LG) test and measuring break-up time (BUT) according to the methods described in detail

by Kalk et al.(11)

Laboratory assessments

Laboratory assessments included serum biochemical analysis and complete blood cell count.

Levels of immunoglobulins (IgG, IgA, IgM) and IgM-RF were measured by nephelometry.

Numbers of circulating CD19+, CD4+ and CD8+ T cells were quantified by FACSCalibur

flow cytometer using TruCOUNTTM tubes (Becton Dickinson). The absolute number was

determined by comparing cellular events to beads events using CellQuest software (Becton

Dickinson).

Subjective assessments

Patients completed the Multidimensional Fatigue Inventory (MFI)(12) and the SF-36.(13) In

addition, a 100-mm Visual Analogue Scale (VAS) was used for rating oral and ocular sicca

complaints.

Extraglandular manifestations (EGM)

Arthralgia, arthritis, renal involvement, oesophageal involvement (confirmed by oesophageal

scintigraphy), polyneuropathy, Raynaud’s phenomenon, tendomyalgia and vasculitis were

defined as EGM. At each visit, EGM were scored as present or not according to protocol.

Serum sickness

Serum sickness was defined as development of fever, lymph node swelling, purpura, myalgia,

arthralgia, thrombocytopenia and proteinuria, and decrease in complement levels. Serum

sickness-like disease was defined as development of some of the symptoms of serum

sickness.

Sample size

Based on a formal sample size calculation, 30 patients were included, 20 assigned to

rituximab and 10 to placebo. The patients were randomly assigned by the pharmacy

department, using a random-number generator on a computer, to one of the two treatment

arms in a 2:1 ratio (rituximab:placebo) in blocks of three. Investigators (who also provided

care and assessed the outcome variables) and patients were blinded to the assigned study

medication. The code was revealed to the investigators after follow-up of all patients was

completed. Because of the double-blind design, we assumed a 5% rate of false-positive

patients in the placebo group with clinical signs of serum sickness. This resulted in an

obligation to terminate the trial if two patients developed clinical signs of serum sickness

after the first or second infusion within the first nine patients, or if three patients developed

clinical signs of serum sickness after the first or second infusion within the first 29 patients.

If for any reason the protocol was terminated, patients were not replaced.

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Statistical analyses

All data analyses were carried out according to a pre-established plan. To compare

treatment effects in time between the two treatment groups, repeated measurements

ANOVA was performed. To determine whether an improvement had occurred over time

relative to baseline, repeated measurements ANCOVA was performed on change from

baseline data. Statistical analyses performed on secondary endpoints are considered to

be of explorative nature. Therefore no corrections were made for multiple comparisons.

The assumptions on homogeneity were met. If data were not normally distributed, a log-

transformation was performed on the data prior to statistical analysis or a distribution-free

alternative was used.

Results

Patients

Between August 2006 and September 2007, 30 patients were randomly assigned to

treatment (figure 1). Baseline characteristics are summarized in table 1. Six patients used

medication which had to be discontinued before inclusion according to the inclusion

criteria.

Efficacy (table 2)

Salivary gland function

Stimulated whole saliva (figure 2a; primary endpoint) significantly improved in the rituximab

group (p=0.018 at week 5 and p=0.004 at week 12) while values in the placebo group

significantly decreased in accordance with the natural progression of the disease. A significant

difference in the mean change from baseline in the stimulated whole salivary flow between

the groups (p=0.038) was found at week 12. Unstimulated whole salivary flow (figure 2b)

and submandibular/sublingual flow also significantly increased in the rituximab group.

Lachrymal gland function

The LG test showed significant improvement in the rituximab group at weeks 5 to 48,

whereas the Schirmer and BUT tests revealed no significant changes.

Laboratory assessments

B cells were completely depleted in patients treated with rituximab after the first infusion

(figure 2c). No significant changes were found in B cell levels in the placebo group. In the

patient with serum sickness (see safety section below), who received only one infusion of

rituximab, B cells reappeared within 12 weeks after treatment. In the other 19 rituximab-

treated patients, B cells returned within 24 to 48 weeks after treatment, although B cell

levels still had not returned to baseline by week 48. Significant differences in the mean

change in absolute B cell count from baseline between the groups were found at weeks 5, 12,

24, 36 and 48 (p<0.05). No significant changes were found in CD4+ and CD8+ T cell levels

in either the rituximab or placebo groups. Rheumatoid factor (figure 2d) levels decreased

significantly in the rituximab group over weeks 5 to 36, and in the placebo group at week 5.

Significant differences in the mean change in rheumatoid factor levels from baseline between

the groups were found at weeks 12, 24 and 36 (p<0.05). The same pattern of change was

found for levels of IgG, IgM and IgA (results not shown).

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Table 1 Patient characteristics. Baseline characteristics of the patients in the placebo and rituximab treat-

ment groups. UWS=unstimulated whole saliva, SWS=stimulated whole saliva.

Variable Placebo (n=10) Rituximab (n=20)

Age (years) 43±17 43±11

Gender 10 females 1 male, 19 females

Disease duration (months) 67±63 63±50

IgG (g/l) 21±7 23±8

IgM-RF (klU/l) 221±245 102±79

Anti-Ro/SSA positive

Anti-La/SSB positive

10 (100%)

8 (80%)

20 (100%)

14 (70%)

Parotid gland swelling 10 (100%) 17 (85%)

UWS, ml/minute* 0.06±0.09 0.17±0.19

SWS, ml/minute 0.42±0.26 0.70±0.57

Extraglandular manifestations

Arthralgia

Arthritis

Renal involvement

Oesophageal involvement

Peripheral polyneuropathy

Raynaud’s phenomenon

Tendomyalgia

Vasculitis

Thyroid dysfunction

5 (50%)

0 (0%)

0 (0%)

1 (10%)

0 (0%)

6 (60%)

8 (80%)

3 (30%)

0 (0%)

15 (75%)

6 (30%)

2 (10%)

0 (0%)

1 (5%)

11 (55%)

17 (85%)

6 (30%)

1 (5%)

Use of artificial tears

Use of artificial saliva

8 (80%)

2 (20%)

14 (70%)

2 (10%)

*Significant difference (p<0.05) between placebo and rituximab group.

Subjective assessments

MFI and SF-36 scores showed the strongest improvements in the rituximab group (figures

2e, 2f). Between the two treatment groups, a significant change was found for the MFI score

for reduced activity (p=0.023) at week 36, the MFI score for reduced motivation (p=0.039)

at week 12 and the SF-36 score for vitality (p=0.013) at week 36. Moreover, all VAS scores

improved in the rituximab group (figures 2g, 2h; table 2), while scores in the placebo group

only showed a significant improvement at week 5. Significant differences in VAS scores

between the groups were seen for dry mouth during the night (p<0.05) at weeks 24, 36 and

48, and dry eyes (p<0.05) at weeks 36 and 48.

Extraglandular manifestations

At baseline there were no differences between the rituximab and placebo group (figure

2i) . The number of reported EGM (absent or present) signif icantly decreased in the

rituximab group compared to placebo for tendomyalgia at weeks 12 and 36 (p=0.029)

and for vasculitis at week 24 (p=0.030). In addition, there was a strong tendency towards

a decrease in the number of reported complaints of Raynaud’s phenomenon (p=0.057),

tendomyalgia (p=0.074) and arthralgia (p=0.058) at week 24. Six patients in the rituximab

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Table 2 Results of laboratory, functional and subjective assessments for the placebo and rituximab treatment groups (mean±SD (median)) at the assessed time-points.

UWS=unstimulated whole saliva, SWS=stimulated whole saliva.

Variable BaselinePlaceboRituximab

Week 5PlaceboRituximab

Week 12PlaceboRituximab

Week 24PlaceboRituximab

Week 36PlaceboRituximab

Week 48PlaceboRituximab

UWS, ml/minute $ + 0.06±0.09 (0.03)0.17±0.19 (0.08)

0.09±0.07 (0.08)0.24±0.22 (0.20)*

0.05±0.05 (0.04)0.23±0.22 (0.19)*

0.08±0.08 (0.09)0.22±0.25 (0.14)

0.07±0.09 (0.02)0.16±0.15 (0.11)

0.05±0.04 (0.04)*0.18±0.18 (0.13)

SWS, ml/minute $ 0.42±0.26 (0.36)0.70±0.57 (0.47)

0.41±0.24 (0.37)0.84±0.71 (0.48)*

0.28±0.17 (0.25)*0.87±0.87 (0.56)*

0.36±0.28 (0.24)0.74±0.60 (0.52)

0.29±0.18 (0.26)*0.64±0.58 (0.44)

0.28±0.21 (0.22)*0.66±0.71 (0.42)

Schirmer’s, mm/5 minutes $ 7±9 (3)11±11 (7)

7±11 (4)10±9 (10)

6±5 (5)11±10 (11)

8±8 (6)12±12 (5)

7 ±7 (5)11 ±10 (7)

5±5 (6)*10±11 (7)

Lysamine green $ 4±1 (4)3±2 (4)

5±1 (5)3±2 (3)*

4±2 (4)3±2 (3)*

4±2 (4)2±2 (2)*

4 ±2 (4)2 ±2 (2)*

4±2 (4)2±3 (1)*

Tear break up time, seconds $ +

3±2 (3)6±2 (6)

3±1 (3)6±3 (6)

3±2 (3)5±3 (5)

5±2 (6)*6±3 (7)

5 ±3 (5)*7 ±3 (8)*

4±3 (4)6±3 (8)

B cells, 109/l $ 0.27±0.12 (0.28)0.21±0.17 (0.18)

0.20±0.09 (0.17)*0.00±0.00 (0.00)*

0.25±0.10 (0.27)0.01±0.03 (0.00)*

0.28±0.11 (0.26)0.05±0.08 (0.03)*

0.28 ±0.12 (0.31)0.10 ±0.08 (0.08)*

0.33±0.15 (0.37)0.17±0.10 (0.15)*

IgM-R, klU/l $ 221±245 (108)102±79 (83)

162±175 (96)*55±36 (53)*

156±138 (102)44±30(36)*

258±260 (113)45±34 (32)*

253 ±256 (119)71 ±68 (54)*

225±199 (126)103±103 (72)

MFI, general fatigue 14±5 (17)16±4 (18)

11±5 (12)*15±4 (16)

13±5 (14)13±4 (13)*

12±5 (12)13±4 (12)*

14 ±4 (14)14 ±4 (14)

14±6 (17)15±4 (16)

SF-36 total 64±17 (65)52±20 (53)

70±17 (70)56±18 (52)

67±15 (71)63±15 (65)*

72±16 (82)67±16 (70)*

63 ±16 (65)60 ±17 (64)*

62±17 (62)55±18 (55)

VAS oral dryness 59±28 (62)55±28 (61)

50±28 (53)47±27 (53)*

53±30 (60)40±27 (40)*

64±27 (74)34±27 (46)*

68 ±26 (79)51 ±28 (61)*

69±25 (76)50±28 (53)*

VAS dry eyes 65±27 (63)59±29 (68)

55±28 (52)49±28 (51)*

61±25 (54)48±29 (47)*

68±24 (74)41±28 (43)*

70 ±27 (72)46 ±27 (52)*

76±19 (80)46±28 (55)*

(mean±SD (median). $no normal distribution. *P<0.05 versus baseline in the same patient group, by ANCOVA analysis, + significant difference (p<0.05) between placebo

and rituximab group at baseline. Bold: comparison of change from baseline between the placebo and rituximab group, by ANOVA analysis, results in a significant

difference (p<0.05) Italic: comparison of change from baseline between the placebo and rituximab group, by ANOVA analysis, results in a difference (p>0.05 and p<0.10).

Due to missing data, the differences between means in this table differ slightly from the means of differences as displayed in the figures.

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group had complaints of arthritis at baseline; this resolved in four patients during follow-

up. In the placebo group, no patients had symptoms of arthritis at baseline; however, three

patients developed symptoms during follow-up. One patient with decreased thyroid function

before rituximab treatment showed a normalization of thyroid function without additional

thyrostatic supplementation. Renal function (two patients had renal tubular acidosis; both

were treated with rituximab) remained stable during follow-up. Clinical symptoms of

polyneuropathy (one patient; rituximab) improved after 12 weeks of follow-up.

Safety (table 3)

Serum sickness

One diabetic patient developed a mild serum sickness-like disease, 14 days after the first

infusion. She developed fever, purpura on both legs and arthralgia. She was admitted

to hospital in order to control her serum glucose levels during administration of i.v.

corticosteroids and non-steroidal anti-inflammatory drugs, and recovered completely in

a few days without developing human anti-chimeric antibodies. The second infusion was

not administered. This patient had not been treated with any immunosuppressive drug

Assessed for eligibility (n=61)

Randomised (n=30)

Excluded (n=31)

Not meeting inclusion criteria (n=7)

Refused to participate (n=24)

Rituximab Placebo

Allocated to intervention (n=20)

Received intervention (n=20)

Did not receive intervention (n=0)

Allocated to intervention (n=10)

Received intervention (n=10)

Did not receive intervention (n=0)

Lost to follow-up (n=0)

Discontinued intervention because

development of serum sickness (n=1)

Lost to follow-up for unknown reasons

after week 12 (n=1)

Discontinued intervention (n=0)

Analysed (n=20)

Excluded from analysis (n=0)

Analysed (n=10)

Excluded from analysis (n=0)

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last available sialometry, IgG, anti-SSA and anti-SSB positivity and rheumatoid factor data.

Figure 1

Patient flow. Out of a cohort of 300 patients, a preselection was made of 61 patients based on last available

sialometry, IgG, anti-SSA and anti SSB positivity and rheumatoid factor data.

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Figure 2

Figures A, B, D, E, F, G, H and I are depicted as mean values of absolute change from baseline. Figure C is

depicted as mean values of absolute numbers. (*) indicate significant (p<0.05) differences within the groups

compared with baseline.

A Stimulated whole saliva (primary endpoint)

B Unstimulated whole saliva

C B cells

D Rheumatoid factor

E MFI score for general fatigue

F SF-36 total score

G VAS oral dryness

H VAS eye dryness

I Extraglandular manifestations

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previously, while none of the 6 patients who had discontinued immunosuppressive drugs 1

to 6 months prior to rituximab treatment developed serum sickness-like disease.

Infections

Twelve of the infections were reported by eleven patients in the rituximab group; four

patients in the placebo group reported a total of seven infections. The rates of infection

were 76 and 65 events per 100 patient-years for the placebo and rituximab groups,

respectively. None of the infections required hospitalization. No opportunistic infections

were seen.

Discussion

This study showed that rituximab-induced B cell depletion can be considered an effective

and safe treatment modality for patients with pSS. B cell depletion resulted in improvement

of objective and subjective parameters of disease activity in pSS patients for at least 6 to 9

months. Amongst others, salivary function improved, fatigue diminished and the number of

EGM was reduced.

Rituximab has already been shown to be a safe and effective treatment for rheumatoid

arthritis (RA) resulting in a decrease in disease activity, diminished radiological progression

and an improved quality of life.(14-16) Previously, the utility of rituximab for the treatment

of SS had only been investigated in a few open-label, Phase II studies and one randomized,

double-blind, placebo-controlled study. Results from open-label studies in terms of objective

and subjective variables were promising(2;3) as was the improvement of systemic features.

(17) Although the duration of treatment effect differed between the trials, in all trials a

significant effect occurred 12 to 24 weeks after treatment. In the randomized, double-blind,

placebo-controlled study of rituximab treatment of SS, a significant improvement in fatigue

(primary endpoint) was noted compared with baseline data in the rituximab group, but

there were no significant changes in secondary endpoints assessing glandular manifestations

(unstimulated salivary flow, Schirmer test).(18) Moreover, this study by Dass et al.(18)

used a less accurate objective eye test (Schirmer test); the Rose Bengal score and LG are

considered to be more accurate.(11) This fact together with the small number of patients

included in the trial (eight rituximab, nine placebo), might explain the lack of significant

improvement in glandular manifestations following rituximab treatment.

In our trial, most significant improvements in endpoints associated with rituximab

treatment were observed between 12 and 36 weeks following treatment. By contrast,

improvement of most of the variables observed in patients in the placebo group occurred

5 weeks after the first infusion. We hypothesize that the improvements observed after

placebo treatment are related to the prednisolone these patients had received before and

during the days after the infusions, although data are inconclusive regarding the effect of

prednisolone on SS symptoms. Although one study reported a significant increase in whole

saliva during the use of low-dose prednisolone,(19) other studies noted no significant

improvement in glandular function.(20;21)

Stimulated whole saliva provides a general indication of overall salivary glandular function,

which is an important outcome in a disease that specifically affects salivary glands. Pijpe et

al. (3) reported a significantly increase of stimulated whole saliva in rituximab treated pSS

patients whose stimulated salivary flow rate was >0.10 ml/minute at baseline. These patients

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also showed significant improvement of subjective parameters as mouth dryness, arthralgia,

physical functioning, vitality and most domains of the MFI. In other words, patients with

some residual secretory potential may benefit the most from rituximab treatment. The

secretory potential at baseline might even discriminate between patients that are considered

to be good responder to rituximab treatment or not. Therefore, stimulated whole saliva

was chosen as the primary endpoint of our study. As cut off value, a stimulated whole saliva

flow rate ≥0.15 ml/min was chosen as this is a flow rate that discriminates between patients

showing disease activity (e.g., progressive loss of secretory function) and patients with an

end stage pSS.(21)

We observed an increase in salivary flow in the rituximab group that exceeded the

intra-patient variability observed for repeated collections of saliva.(8) This increase is also

reflected by the improvements of subjective scores for dry mouth and indicates that these

changes are clinically meaningful for the patients. The, non-significant, baseline difference

between the groups for the flow rate of stimulated whole saliva was caused by high salivary

flow rates before inclusion in a few patients. All patients in the study were required to have

a stimulated whole saliva flow ≥0.15 ml/min. This meant that all patients had a clinically

relevant functional secretory salivary gland capacity. Our pilot study revealed that no

relevant improvement in salivary gland function can be expected in patients with little or no

secretory potential at baseline.

In RA clinical trials of rituximab, the number of reported (serious) infections and infusion

reactions is within the range expected for patients with RA treated with biological agents.

Therefore, the risk:benefit ratio is considered to be good regarding rituximab treatment

of RA.(22) In clinical trials of rituximab treatment of other autoimmune diseases (including

SS), reported numbers of infusion reactions and infections vary widely; this is possibly due

to variability in how these adverse events are defined or to small patient numbers. The

incidence of infusion reactions and infections reported for the rituximab group in this trial

was largely comparable to that of the placebo group and was lower or within the same range

Table 3 Adverse events observed in patients following treatment with rituximab or placebo.

Events Placebo (n=10)

Rituximab (n=20)

Early infusion reaction 0 2 (10%)

Late infusion reaction 0 2 (10%)

Serum sickness 0 1 (5%)

Infections within 2 weeks after

infusion

Upper airway infection 0 1 (5%)

Parvovirus 0 1 (5%)

Infections during 48 weeks

of follow-up

Otitis media 0 2 (10%)

Upper airway infection 4 (40%) 4 (20%)

Recurrence of ocular toxoplasmosis 0 1 (5%)

Parotid gland infection 0 3 (15%)

Recurrence of herpes zoster 1 (10%) 0

Epstein-Barr virus 1 (10%) 0

Rubella 1 (10%) 0

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as that reported previously.(23) Also, the rate of infections per 100 patient-years is lower

compared with the rate in RA patients treated with rituximab. This might be explained by

the fact that our patients did not use any other immunosuppressive therapy.(24)

When compared to lymphoma patients, RA patients and systemic lupus erythematosus

(SLE) patients treated with rituximab, patients with pSS develop serum sickness(-

like) disease more frequently (6% to 27%).(25) A therapy-related explanation for this

phenomenon might be that usually higher doses of steroids and/or other immunosuppressive

drugs besides rituximab have been or are given to RA and SLE patients, whereas our

pSS patients received no other co-medication than a 5 days period of steroids after i.v.

administration of rituximab. Another therapy-related explanation is that RA and SLE patients

often have been treated with intensive immunosuppressive regiments including biological

agents before they were subjected to rituximab treatment, whereas our pSS patients are

far more medication-naïve at the time of rituximab treatment. The higher susceptibility for

serum sickness could also be inherent to the disease itself. The pSS patients in this trial,

as well as in our pilot trial, (3) who developed serum sickness were more likely to have

an active, early and progressive form of SS. It is possible that such pSS patients are more

prone to develop serum sickness. Furthermore, hypergammaglobulinemia is common in

pSS patients, which could make these patients prone to the development and deposition of

immune complexes and thus to serum sickness(-like) disease.(18)

Because of the higher risk of developing serum sickness(-like) disease in SS patients, we

decided to increase the steroid dose. Only one patient in the current study developed serum

sickness-like disease (5%), which is considerably lower than the incidence reported in our

open-label study (27%).(3) Based on these findings, we would recommend administering 100

mg methylprednisolone immediately prior to each infusion of rituximab. The oral regimen of

prednisolone in the days following each infusion is a point of interest and should be explored

in future trials. The administration of higher doses of prednisolone in the days following

infusion, such as is performed during lymphoma treatment, should also be considered.

This study indicates that rituximab treatment could be effective for pSS patients with

active disease and remaining salivary gland secretory potential as well as for pSS patients

with EGM. Future trials with rituximab in pSS are warranted with inclusion of larger groups

of patients and with defining less strict inclusion criteria (e.g., no restriction to salivary

gland function ≥ 0.15 ml/min and auto-antibody positivity) in order to be able to extrapolate

the results to a larger group of pSS patients. Besides inclusion criteria, attention should be

given to defined criteria for response to treatment. Activity scores for pSS have now been

developed and wait for validation. These scores should be included in response criteria to

be used in future trials.

Based on the promising results of this study and on our study on retreatment with

rituximab, which resulted in a beneficial effect comparable to that of the first treatment with

this biological (26), a maintenance therapy with rituximab infusions every 6 to 9 months

may be a reasonable approach. Advantages of maintenance therapy might be a reduction or

even arrest of disease progression and improvement of quality of life for a long period. This

improvement will be a great achievement in SS patients, as SS has a large impact on health-

related quality of life, employment and disability.(1) A threat might be the, so far unknown,

long-term side effects of repeated B cell depletion. The timing of retreatment could be

based on return of symptoms, however, retreatment just before return of symptoms would

even be better.

In conclusion, this study indicates that rituximab could be an effective and safe treatment

Ch

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116

modality for patients with pSS. B cell depletion resulted in improvement of the primary

endpoint stimulated whole saliva. Explorative analyses also showed improvements

for at least 6 to 9 months’ duration of objective and subjective secondary endpoints of

disease activity. As pSS has a great impact on health-related quality of life, employment

and disability(1), it is worthwhile to further explore the role of rituximab in a large size

randomized controlled trial.

Acknowledgements

We are grateful to Janita Kuiper, Philip M Kluin, Jaqueline E van der Wal, Khaled Mansour,

Gustaaf W van Imhoff and Justin Pijpe for their support and meaningful discussions.

This investigator-driven study was financially supported by Roche, Woerden, The

Netherlands, which also supplied study medication. There was no involvement of this funding

source in study design, patient recruitment, data collection, analysis and interpretation and

writing of the report. Statistical analyses were performed by the statistical department

of Xendo Drug Development BV., Groningen, The Netherlands, which is an independent

contract research organization.

Medical writing support was provided by Adelphi Communications during the final

preparation of this article, supported by F. Hoffmann-La Roche Ltd.

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ent

117

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(2) Devauchelle-Pensec V, Pennec Y, Morvan J, Pers JO, Daridon C, Jousse-Joulin S et al. Improvement

of Sjögren’s syndrome after two infusions of rituximab (anti-CD20). Arthritis Rheum 2007;

57(2):310-7.

(3) Pijpe J, van Imhoff GW, Spijkervet FKL, Roodenburg JLN, Wolbink GJ, Mansour K et al. Rituximab

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(4) Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE et al. Classification

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(5) Pijpe J, Kalk WWI, van der Wal JE, Vissink A, Kluin PM, Roodenburg JLN et al. Parotid gland

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(6) Dawes C. Circadian rhythms in human salivary flow rate and composition. J Physiol 1972; 220(3):529-

45.

(7) Ferguson DB, Fort A, Elliott AL, Potts AJ. Circadian rhythms in human parotid saliva flow rate and

composition. Arch Oral Biol 1973; 18(9):1155-73.

(8) Burlage FR, Pijpe J, Coppes RP, Hemels MEW, Meertens H, Canrinus A et al. Accuracy of collecting

stimulated human parotid saliva. Eur J of Oral Sci 2005; 113(5):386-90.

(9) Kalk WW, Vissink A, Stegenga B, Bootsma H, Nieuw Amerongen AV, Kallenberg CG. Sialometry and

sialochemistry: a non-invasive approach for diagnosing Sjögren’s syndrome. Ann Rheum Dis 2002;

61(2):137-44.

(10) Kalk WWI, Vissink A, Spijkervet FKL, Bootsma H, Kallenberg CGM, Nieuw Amerongen AV.

Sialometry and sialochemistry: diagnostic tools for Sjögren’s syndrome. Ann Rheum Dis 2001;

60(12):1110-6.

(11) Kalk WW, Mansour K, Vissink A, Spijkervet FK, Bootsma H, Kallenberg CG et al. Oral and ocular

manifestations in Sjögren’s syndrome. J Rheumatol 2002; 29(5):924-30.

(12) Smets EM, Garssen B, Bonke B, De Haes JC. The Multidimensional Fatigue Inventory (MFI)

psychometric qualities of an instrument to assess fatigue. J Psychosom Res 1995; 39(3):315-25.

(13) Ware JE, Jr., Sherbourne CD. The MOS 36-item short-form health survey (SF-36). I. Conceptual

framework and item selection. Med Care 1992; 30(6):473-83.

(14) Cohen SB, Emery P, Greenwald MW, Dougados M, Furie RA, Genovese MC et al. Rituximab for

rheumatoid arthritis refractory to anti-tumor necrosis factor therapy: Results of a multicenter,

randomized, double-blind, placebo-controlled, phase III trial evaluating primary efficacy and safety

at twenty-four weeks. Arthritis Rheum 2006; 54(9):2793-806.

(15) Mease PJ, Revicki DA, Szechinski J, Greenwald M, Kivitz A, Barile-Fabris L et al. Improved health-

related quality of life for patients with active rheumatoid arthritis receiving rituximab: Results

of the Dose-Ranging Assessment: International Clinical Evaluation of Rituximab in Rheumatoid

Arthritis (DANCER) Trial. J Rheumatol 2008; 35(1):20-30.

(16) Popa C, Leandro MJ, Cambridge G, Edwards JC. Repeated B lymphocyte depletion with rituximab

in rheumatoid arthritis over 7 yrs. Rheumatology (Oxford) 2007; 46(4):626-30.

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(17) Gottenberg JE, Guillevin L, Lambotte O, Combe B, Allanore Y, Cantagrel A et al. Tolerance and short

term efficacy of rituximab in 43 patients with systemic autoimmune diseases. Ann Rheum Dis 2005;

64(6):913-20.

(18) Dass S, Bowman SJ, Vital EM, Ikeda K, Pease CT, Hamburger J et al. Reduction of fatigue in Sjögren’s

syndrome with rituximab: results of a randomized, double-blind, placebo controlled pilot study.

Ann Rheum Dis 2008; 67(11):1541-4.

(19) Miyawaki S, Nishiyama S, Matoba K. Efficacy of low-dose prednisolone maintenance for saliva

production and serological abnormalities in patients with primary Sjögren’s syndrome. Intern Med

1999; 38(12):938-43.

(20) Fox PC, Datiles M, Atkinson JC, Macynski AA, Scott J, Fletcher D et al. Prednisone and piroxicam for

treatment of primary Sjögren’s syndrome. Clin Exp Rheumatol 1993; 11(2):149-56.

(21) Pijpe J, Kalk WWI, Bootsma H, Spijkervet FKL, Kallenberg CGM, Vissink A. Progression of salivary

gland dysfunction in patients with Sjögren’s syndrome. Ann Rheum Dis 2007; 66(1):107-12.

(22) Fleischmann RM. Safety of Biologic Therapy in Rheumatoid Arthritis and Other Autoimmune

Diseases: Focus on Rituximab. Semin Arthritis Rheum 2008.

(23) Gurcan HM, Keskin DB, Stern JN, Nitzberg MA, Shekhani H, Ahmed AR. A review of the current use

of rituximab in autoimmune diseases. Int Immunopharmacol 2009; 9(1):10-25.

(24) Keystone E, Fleischmann R, Emery P, Furst DE, van Vollenhoven R, Bathon J et al. Safety and efficacy

of additional courses of rituximab in patients with active rheumatoid arthritis: an open-label

extension analysis. Arthritis Rheum 2007; 56(12):3896-908.

(25) Meijer JM, Pijpe J, Bootsma H, Vissink A, Kallenberg CG. The future of biologic agents in the

treatment of Sjögren’s syndrome. Clin Rev Allergy Immunol 2007; 32(3):292-7.

(26) Meijer JM, Pijpe J, Vissink A, Kallenberg CG, Bootsma H. Treatment of primary Sjögren syndrome

with rituximab: extended follow-up, safety and efficacy of retreatment. Ann Rheum Dis 2009;

68(2):284-5.

Rituximab treatment119

Jiska M Meijer1, Stefan O Schonland2, Giovanni Palladini3,

Giampaolo Merlini3, Ute Hegenbart2, Olga Ciocca4, Vittorio

Perfetti3, Martha K Leijsma5, Hendrika Bootsma5, Bouke PC

Hazenberg5

Arthritis Rheum. 2008 Jul;58(7):1992-9

Chapter 6

Sjögren’s syndrome and localized

nodular cutaneous amyloidosis:

coincidence or a distinct clinical entity?

Departments of 1Oral and Maxillofacial Surgery and 5Rheumatology and Clinical Immunology,

University Medical Center Groningen, University of Groningen, The Netherlands; Department

of 2Hematology, Oncology and Rheumatology, University of Heidelberg, Germany; 3Amyloid

Center, Biotechnology Research Laboratories and 4Department of Dermatology Fondazione

IRCCS Policlinico San Matteo and University of Pavia, Italy

Ch

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Abstract

Objective To report 8 patients with Sjögren’s syndrome (SS) and localized nodular cutaneous

amyloidosis and to examine serologic and immunohistologic findings that may link the 2

diseases.

Methods The databases of 3 amyloidosis centers were searched for patients with localized

nodular cutaneous amyloidosis and SS. Eight patients with this combination were identified,

and clinical, serologic and histologic parameters were retrospectively evaluated.

Results Among the 8 patients with a clinical diagnosis of SS, 6 fulfilled the American-european

Consensus Group criteria for SS. All of the patients were women in whom SS had been

diagnosed at a median age of 47 years (range 30-61 years) and amyloid had been diagnosed

at a median age 60 years (range 42-79 years). The presence of the immunoglobulin light

chain type of amyloid (AL) was confirmed in 4 patients. In 3 of these 4 patients as well as 2

other patients, a light chain-restricted plasma cell population was observed near the amyloid

deposits. Progression to systemic amyloidosis was not observed in any patient during a

median follow-up of 3.5 years.

Conclusions SS should be considered in patients with cutaneous amyloidosis. The combination

of cutaneous amyloid and SS appears to be a distinct disease entity reflecting a particular

and benign part of the polymorphic spectrum of lymphoproliferative diseases related to SS.

Sjö

gren

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d am

yloi

dosi

s123

Introduction

Sjögren’s syndrome (SS) is a chronic lymphoproliferative autoimmune disease characterized

by disturbances in T lymphocytes, B lymphocytes and exocrine glandular cells.(1) SS can be

primary or secondary, with the latter entity being associated with another autoimmune

disease such as rheumatoid arthritis or systemic lupus erythematosus. Lymphocytic

infiltrates, consisting of T lymphocytes and B lymphocytes, are a characteristic histopatho-

logic f inding in SS. The presence of autoantibodies and hypergammaglobulinemia is

considered to reflect polyclonal B lymphocyte hyperactivity. Systemic complications of SS

are associated with this polyclonal B lymphocyte hyperactivity and with the development of

clonal B lymphocyte proliferation. As a reflection of the latter, a malignant B cell lymphoma

develops in ~5% of patients with SS.(2)

Amyloidosis refers to a variety of protein-folding diseases caused by extracellular

deposition of amyloid fibrils. The peptide subunit of the protein fibrils varies among the

different types of amyloidosis and is the basis for the current chemical classification.(3) AL

amyloid (formerly called primary amyloid) refers to the immunoglobulin light chain-

associated amyloid, and AA amyloid (formerly called secondary amyloid) refers to the

inflammation-associated amyloid. The diagnosis of amyloidosis is based on the characteristic

apple-green birefringence under polarized light of a biopsy specimen stained with Congo

red.(4) Amyloidosis can be divided in systemic and localized forms. In the systemic form,

there is widespread deposition of amyloid in organs and tissues and in the localized form,

amyloid deposition is restricted to one single organ, tissue, or site of the body.(4;5)

Although it is very rare, systemic amyloidosis has been observed in patients with SS;

systemic AA amyloidosis may occur because of longstanding inflammation(6), and systemic

AL amyloidosis may occur because of the development of an immunoglobulin light chain-

producing lymphoproliferative disease.(7) Systemic AL amyloidosis itself can affect the

lacrimal and salivary glands and is therefore one of the causes of sicca syndrome.(7) SS has

also been associated with the presence of localized amyloid in sites such as the lungs(8), the

breast(9), the tongue(10), and the skin.(11) Three types of localized cutaneous amyloidosis

can be recognized: macular, lichen and nodular types, of which the nodular type is the

rarest.(12) Nodular cutaneous amyloidosis has been related frequently to deposition of

immunoglobulin light chains that have been produced by a clonal plasma cell population. The

typical clinical presentation involves single or multiple nodules or plaques on the trunk or

limbs.(11) Deposition of amyloid usually takes place in the dermis, subcutis and associated

blood vessels.(12;13)

The estimated prevalence of SS in the general population is ~0,5-2%.(14;15) Cutaneous

nodular amyloidosis is extremely rare, with ~60 cases reported in the literature.(16)

Despite the rare occurrence of cutaneous amyloidosis, 16 cases have been reported in

patients with SS, and these 16 cases represent ~25% of the reported cases.(11-13) The link

between cutaneous amyloidosis and SS is, however, still unresolved. Here we will discuss the

possible relationship between the 2 entities after reporting clinical, serologic, and histo-

patho logic data for 8 new cases.

Ch

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124

Patients and methods

We retrospectively searched the patient registries for 3 amyloidosis referral centres (a total

of 2306 patients (1421 Italian, 520 Dutch and 365 German patients) , to identify the

combination of cutaneous localized amyloid and SS. Eight patients (0.3%) with this combination

of diseases were identified. The current American-European Concensus Group criteria were

used to determine how many cases adhered to the current definition of SS.(17) Extraglandular

manifestations of SS were defined as the presence or confirmed records of purpura, lung or

neurological involvement, synovitis, myositis, vasculitis, lymphadenopathy, enlarged spleen or

previous lymphoma during the evolution of the disease. Histopathologic data and information

regarding recently determined serologic parameters were collected. The local ethics

committees approved the study, and all patients gave informed consent.

The serologic parameters comprised antinuclear antibodies, extractable nuclear antigens,

SSA antigens, SSB antigens, rheumatoid factor, cryoglobulins, anti-double-stranded DNA, total

protein, gamma globulin, serum amyloid A protein, serum M (monoclonal) protein, serum κ

free light chain, serum λ free light chain, serum κ:serum λ free light chain ratio, alkaline

phosphatase, and creatinine. Kidney function was evaluated by measuring the amount of

proteinuria and the creatinine clearance.

Histopathology reports for biopy specimens obtained from the parotid gland, other salivary

glands, skin and abdominal fat were retrieved; if the biopsy specimens were judged inadequate

for the current purpose, they were reexamined if the original tissue blocks were still available.

Figure 1

A. Cutaneous amyloid papules and nodules located on the lower legs of patient 2

B. Higher-magnification view of papules and nodules shown in A.

C. Cutaneous amyloid papules and nodules located on the upper front side of the thorax of patient 7

D. Higher-magnification view of papules and nodules shown in C.

Sjö

gren

an

d am

yloi

dosi

s125

Histologic examination focused on whether amyloid was present or absent (as determined by

Congo red staining) in the investigated tissues and whether signs of SS (such as lymphocytic

infiltrates, myoepithelial islands, focus score ≥ 1) were present in the parotid gland or salivary

gland. The skin biopsy specimen was evaluated specifically for the type of amyloid involved, by

using a panel of antibodies directed to amyloid A protein, λ and κ light chains, and trans-

thyretin, and also for the presence of a light chain-restricted plasma cell population located

nearby the amyloid deposits.

Results

The characteristics of the patient are shown in table 1. All of the patients were women. One

patient (patient 6) was of Indonesian descent and the other 7 patients were white. SS had

been diagnosed at a median age of 47 years (range 30-61 years). Amyloid had been detected

at median age 60 years (range 42-79 years): in 3 patients amyloid was detected 18 years, 5

years, and 2 years, respectively, before the diagnosis of SS; in 1 patient, amyloid was

detected simultaneously with the diagnosis of SS; and in 4 patients amyloid was detected 10

years, 29 years, 34 years, and 34 years after the diagnosis of SS. In all 8 patients the

determination that SS had been present for many years was made on a clinical basis. Six of

these eight patients (75%) fulfilled the current strict American-European concensus Group

criteria for SS (see table 2). In the remaining 2 patients all of the criteria could not be

applied, because data about salivary gland involvement (flow and biopsy) were not available.

In the latter 2 cases, however, the diagnosis of SS was very likely on clinical grounds. All

patients had sicca symptoms; 6 patients had xerostomia, and 7 patients had xerophtalmia.

No amyloid deposits were observed in the salivary gland biopsy specimens that were

investigated. Six patients had extraglandular manifestations of SS (see table 1).

The amyloid deposits were generally asymptomatic, sparse, erythematous yellowish or

orange papules and small nodules on the limbs, or, less frequently, the chest or abdomen.

(figure 1) One patient (patient 4) also presented with large, brownish, hyperkeratotic dorsal

patches.

Figure 2

Photomicrographs of a skin biopsy specimen obtained from patient 5, showing Congo red-stained amyloid

deposits in the skin.

A. Normal light shows amyloid staining (arrows).

B. Polarized light shows apple-green birefringence of positively stained amyloid deposits.

(Original magnification 200 x.)

Chapter 6

126

Table 1 Characteristics of the patients*.

Characteristics Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8

Age, years, at diagnosis of Sjögren

(year of diagnosis)57 (1994) 47 (2005) 61 (2000) 47 (1994) 45 (1965) 44 (1970) 30 (1975) 61 (2006)

Age, years, at detection of amyloid

(year of detection)67 (2004) 44 (2003) 43 (1982) 42 (1989) 79 (1999) 79 (2004) 59 (2004) 61 (2006)

Sicca symptoms XerophthalmiaXerophthalmia,

xerostomia

Xerophthalmia,

xerostomia

Xerostomia Xerostomia,

xerophthalmia

Xerostomia,

xerophthalmia

Xerostomia,

xerophthalmia

Xerophthalmia

Extraglandular manifestationsFatigue, arthralgia,

Raynaud’s

Fatigue, arthralgia,

Raynaud’s

Fatigue, arthralgia CREST syndrome,

fatigue

Fatigue Fatigue, arthralgia,

Raynaud’s

Serology:SAA, mg/liter (normal <4.3) 7.3 8.3 3.7 15.7 1.0 5.0 1.0 ND

Antinuclear antibodies: pos pos pos neg pos pos pos pos

IgM-Rf, IU/ml (normal < 15) ND ND pos ND 76 75 188 Pos

total protein, mg/dl (normal 6.5-7.9) 6.3 10.2 7.2 7.4 7.0 7.9 9.3 8.8

Gamma globulin, mg/dl (normal 0.7-3.0) 0.9 4.17 1.39 1.07 1.49 1.54 3.97 3.31

κ FLC level, mg/liter (normal 3.3-19.4) † 12.4 64.7‡ ND 20.9 19.4 18.5 70.3 43.1

λ FLC level, mg/liter (normal 5.7-26.3) † 12.3 16.6 ND 8.9 45.9‡ 23.2 41.1 52.7

FLC ratio (normal 0.26-1.65) † 1.01 3.90‡ ND 2.34‡ 0.42 0.80 1.71§ 0.82

Pathology:Salivary gland biopsy ND SS ND SS SS ND SS (parotid) ND

Skin

Congo red staining

Immunohistology

Predominant plasma cells

Pos

ND

ND

Pos

AL κ ¶

λ

Pos

AL λ ¶

No plasma cells

Pos

ND

ND

Pos

AA neg, AL λ pos

λ

Pos

AA neg

λ

Pos

AA neg

κ

Pos

AL κκ

BMPCs, (%) ND 5 ND ND 3 ND 1 9

Location of amyloid Left leg Arm, back, legs abdomen Back Legs Legs Arms, legs, trunk abdomen

Disease progression:Local cutaneous progression /

new cutaneous sitesYes / yes No / yes No / no No / no Yes / yes Yes / no Yes / yes #

Systemic amyloidosis No No No No No No No No

Treatment and medicationMethylprednisolone

4 mg/dayNo No No

Surgical excision,

electrocoagulationNo No Surgical excision

* All patients were women. SS= Sjögren’s syndrome (for salivary gland biopsy, SS=hitologic changes speciic for SS); Raynaud’s = Raynaud’s phenomenon; CREST syndrome = calcinosis, Raynaud,

esophagial dysmotility, sclerodactily, telangiectasis; SAA = serum amyloid A protein; ND = Not Done; IgM Rf = IgM Rheumatoid factor; FLC = free light chain; AL = amyloid light chain, BMPCs =

bone marrow plasma cells. † reference intervals according to Katzmann et al.(21) ‡ indication of clonal light chain overproduction. § levels of both λ and κ are increased, but the level of κ is more

increased than that of λ, resulting in an increased ratio. ¶ Invetigated using immunoelectronmicroscopy. # till slowly progressive in terms of the number and size of the lesions.

Sjögren and amyloidosis127

In 6

patie

nts th

e am

yloid

was th

ough

t to b

e o

f type A

L: in

4 o

f these

patie

nts, am

yloid

was p

ositive

for a p

articular ligh

t chain

by im

muno

ele

ctron m

icrosco

py, an

d in

3 o

f these

4

patie

nts as w

ell as 2

oth

er p

atients, a ligh

t chain

-restric

ted p

lasma ce

ll po

pulatio

n w

as

obse

rved in

close

pro

xim

ity to th

e am

yloid

depo

sits in th

e sk

in. (figu

res 2

and 3

) In th

e

rem

ainin

g 2 p

atients im

muno

histo

logic an

alysis was n

ot p

ossib

le, b

ecau

se m

aterial h

ad n

ot

been o

btain

ed fo

r that p

urp

ose

. Bio

psy sam

ple

s of su

bcu

taneo

us fat d

id n

ot sh

ow

any

amylo

id in

7 p

atients an

d w

ere n

ot o

btain

ed in

1 p

atient (p

atient 8

).

Sym

pto

ms o

f syste

mic

AL a

mylo

ido

sis were

ab

sen

t in a

ll patie

nts. In

partic

ula

r,

ech

ocard

iogram

s were

no

rmal, th

ere

was n

o sign

ificant p

rote

inuria, an

d re

sults o

f seru

m

creatin

ine an

d live

r functio

n te

sts were

with

in re

fere

nce

limits in

all patie

nts. N

one o

f the

patie

nts h

ad B

ence

Jones p

rote

inuria o

r a seru

m M

pro

tein

, and re

sults o

f imm

uno

fixatio

n

studie

s of se

rum

and u

rine w

ere

negative

in all p

atients. S

eru

m im

muno

globulin

free ligh

t

chain

conce

ntratio

ns w

ere

measu

red in

7 p

atients (tab

le 1

). The κ

: λ ratio

was ab

ove

the

refe

rence

range

in 3

patie

nts (p

atients 2

, 4 an

d 7

), and 1

patie

nt (p

atient 5

) had

an in

crease

in th

e le

vel o

f λ fre

e ligh

t chain

desp

ite a n

orm

al κ: λ

ratio. T

he typ

e o

f amylo

id (in

patie

nts

2 an

d 5

) and th

e typ

e o

f pre

do

min

ant p

lasma ce

lls in th

e sk

in b

iopsy (in

patie

nts 2

, 5 an

d 7

)

corre

spo

nded to

the typ

e o

f circulatin

g free ligh

t chain

s with

conce

ntratio

ns ab

ove

the

upper re

fere

nce lim

it (figure 4

). In p

atient 4

, the typ

e of am

yloid

was n

ot ch

aracterize

d.

The m

edian

follo

wup w

as 3.5

years (ran

ge 1-2

5 ye

ars) after th

e d

iagno

sis of am

yloid

osis.

Lo

calized am

yloid

rem

ained stab

le fo

r years o

r show

ed o

nly m

ino

r cutan

eo

us p

rogre

ssion.

Pro

gressio

n to

system

ic AL am

yloid

osis w

as no

t seen in

any o

f the p

atients. N

o clin

ical

relevan

t com

orb

idity w

as obse

rved in

any o

f the p

atients.

Disc

ussio

n

The cu

rrent re

sults p

rovid

e su

ppo

rt for th

e h

ypo

thesis th

at cutan

eo

us n

odular am

yloid

osis

in S

S is a distin

ct clinical e

ntity. A

mylo

id d

erive

d fro

m im

muno

globulin

light ch

ains (typ

e AL)

is locally p

rod

uce

d b

y a light c

hain

-restric

ted p

lasma ce

ll po

pulatio

n in

the sk

in. T

his

Fig

ure

3

Imm

uno

pero

xid

ase-stain

ed skin

bio

psy sp

ecim

en o

btain

ed fro

m p

atient 5

, show

ing am

yloid

deposits in

the

skin w

ith p

lasma ce

lls nearb

y.

A. M

any p

lasma ce

lls imm

uno

reactive

for λ

light ch

ain, lo

cated in

close

pro

xim

ity to λ

-po

sitive am

yloid

de-

posits.

B. P

lasma ce

lls imm

uno

reactive fo

r κ ligh

t chain

and κ

-negative am

yloid

deposits.

(Origin

al magn

ificatio

n 2

00 x

)

κ

λ

κλ

λ λλ λ κ

κκ

λ κ κ

λ

Ch

apte

r 6

128 hypothesis concerning a distinct disease entity has been based on 4 related issues, as

follows: localized deposition of AL amyloid, the type of AL amyloid involved, the presence of

light chain-restricted plasma cells near the amyloid deposits, and the relationship with SS.

Systemic amyloidosis was not detected in any of our patients nor in the patients

described in the literature, and no evidence of systemic amyloidosis was observed in any

patient during followup. Therefore, localized deposition of amyloid is thought to be present

in all these patients.

In 6 patients, the amyloid was characterised to be type AL, by immunohistology of

amyloid itself or by the presence of a light chain-restricted plasma cell population found near

the amyloid deposits. This finding strongly supports the light chain origin of this amyloid. It

should be noted that detection of AL amyloid by immunohistology is frequently (~32-35% of

patients) negative because of lack of reactivity with the antibodies used.(18;19) In the

patients with cutaneous nodular amyloidosis and SS described in the literature, only type AL

has been detected, when typing of amyloid was possible.(11) Therefore, it is likely that AL

amyloid was the actual type all 8 patients.

Table 2 Characteristics of the patients based on the American-European Concensus group revised criteria for

Sjögren’s Syndrome. *

Patient 1

Patient2

Patient 3

Patient 4

Patient 5

Patient 6

Patient 7

Patient 8

1. Ocular symptoms Yes Yes Yes Yes Yes Yes Yes Yes

2. Oral symptoms Yes Yes Yes Yes Yes Yes Yes Yes

3. Ocular signs (Schirmer and Rose Bengal score)

Yes Yes ND Yes Yes Yes Yes Yes

4. Histopathology ND Yes Yes Yes Yes ND Yes ND

5. Salivary gland involvement ND Yes Yes ND Yes ND Yes ND

6. Autoantibodies to SSA or SSB (SSA/SSB)

No (-/-)

Yes (+/+)

Yes (+/ND)

No (-/-)

Yes (+/-)

No (-/-)

Yes (+/+)

Yes (+/+)

Total score 3 6 5 4 6 3 6 5

Diagnosis of Sjögren’s Syndrome according to the criteria

No Yes Yes Yes Yes No Yes Yes

* According to the American-European Concensus Group criteria (17), primary Sjögren’s syndrome may be

defined as the presence of any 4 out of the 6 following items (including 4 or 6), or any 3 of item 3,4,5 or 6.

Ocular symptoms: a positive response tot at least one question; Have you had daily, persistent, troublesome

eyes for more than 3 months? Do you have a recurrent sensation of sand or gravel in the eyes? Do you use

tear substitutes more than 3 times a day?

Oral symptoms: a positive respons to at least one question; Have you had daily feeling of dry mouth for more

than 3 months? Have you had recurrently or persistent swollen salivary glands as an adult? Do you frequently

drink liquids to aid in swallowing dry food?

Ocular signs: posititve results for at least one test; Schirmer’s test, without anesthesia (≤ 5 mm in 5 minutes)

or Rose Bengal score (≥ 4 van Bijsterveld’s scoring system)

Histopathology: Labial salivary gland: focusscore ≥ 1 or Parotid gland: MESA, myoepithelial islands

Salivary gland involvement: at least one positive test; Unstimulated whole saliva flow (≤ 1.5 ml in 15 minutes)

or Sialectasia on parotid sialography or Abnormal salivary scintigraphy

Autoantibodies to SSA/Ro and/or SSB/La

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In 5 of our patients, the presence of light chain-restricted plasma cells near the amyloid

was actually detected in the skin biopsy specimen, suggesting a possible relationship between

local production of a single free light chain by plasma cells followed by amyloid deposition.

This finding is consistent with the literature.(11;13) In the remaining 3 patients (patients 1, 3

and 4) a search for plasma cells was not performed in the original skin biopsy specimen, and

a specimens were unavailable to allow performance of this search at the time of our

retrospective study.

The situation of plasma cells being in close proximity to the cutaneous epithelium may be

explained by subclinical homing of these cells (or their precursor B lymphocytes) to the skin

as a result of SS itself, which is an autoimmune epithelitis.(15) This explanation is highly

speculative because of the large differences between glandular epithelium and cutaneous

epithelium.

Serum free light chain concentrations are increased in patients with primary SS, especially

those with extraglandular involvement, as compared with healthy control subjects.(20)

However, Gottenberg et al. reported only 3% of patients in whom serum free light chains

were elevated in the same high range as that in 4 patients in the current study who had a

single increased free light chain; i.e., >45 mg/l for λ light chain and >50 mg/l for κ light chain.

(20) In the 4 patients in the current study, the increased serum concentration of a free light

chain might reflect overflow of local intracutaneous production into the systemic circulation.

No M protein in serum, Bence Jones protein in urine, or plasma cell clonality in salivary

glands and bone marrow was observed in the specimens that were studied, and systemic AL

amyloidosis did not develop in any of the patients. Therefore, no cause of the increased light

chain concentration in the blood than the skin seems to be likely. Symptoms of SS usually

develop very gradually, and therefore this syndrome often has not been diagnosed until

years after onset of the first symptoms. In patients 2 and 4, we know that the symptoms

started before amyloidosis was diagnosed; therefore, SS was probably already present

before the development of amyloid. Why amyloid was detected 18 years before SS in the

third patient (patient 3) cannot be explained and this has not been reported previously in

the literature.

Figure 4

Immunoelectron microscopy of a skin biopsy speci-

men obtained from patient 2, showing immunogold

labeling with anti-κ antibodies of amyloid fibrils lo-

cated around the plasma cell and of vacuoles inside

the plasma cell.

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The 4 issues mentioned above may lead to the following hypothesis of a distinct disease

entity: although SS is considered to be a T lymphocyte-mediated disease, its extraglandular

manifestations are associated with an increase in B lymphocyte activity. Cutaneous nodular

amyloidosis in SS seems to be the result of a benign clonal proliferation of plasma cells in the

skin that is part of the spectrum of lymphoproliferative diseases associated with SS. This

disease entity can be distinguished from the other lymphoproliferative diseases of this type

by the differentiation of B lymphocytes to plasma cells and the homing of plasma cells to the

skin, with local production of a single immunoglobulin light chain that is used to be deposited

locally as AL amyloid fibrils.

It is remarkable and puzzling that most lesions develop on the extremities and especially

on the legs; a plausible explanation is lacking. The course of the disease has proven to be

benign in the current cases as well as the cases described in the literature. Treatment

options for nodular localized amyloidosis are therefore limited to local removal of amyloid,

such as surgical excision, cryotherapy, electrodessication and carbon dioxide laser

treatment.(12) In our opinion, treatment is recommended only if there is any discomfort for

the patient or for esthetic reasons.

Apart from the skin, localized nodular AL amyloidosis in SS has also been described

sporadically in the lung(8) and in the breast(9). If these other 2 sites are also consistently

connected to SS, then these 3 different amyloid sites may be grouped together in an even

larger disease entity, i.e., SS-associated localized nodular amyloidosis (for which the acronym

SALNA can be used).

In conclusion, localized nodular cutaneous amyloidosis is very rare and many of the

reported cases are related to SS. Therefore, it is useful to look for signs of SS in patients

with cutaneous amyloidosis. This type of amyloid appears to be related to local production

of one of the free light chains by light chain-restricted plasma cells that had infiltrated the

skin, possibly as part of the autoimmune epithelitis. We hypothesize that this combination

of amyloid and SS is a distinct disease entity reflecting a particular and benign part of the

polymorphic spectrum of lymphoproliferative diseases related to SS.

Acknowledgements

We thank Prof. Dr. Philip Kluin for providing the photographs in figures 2 and 3, Dr. Laura

Verga for providing the photomicropraphs in figure 4, and Johan Bijzet for his technical as-

sistance.

This study is part of the EURAMY project 037525 that is supported by funding of the

Sixth Research Framework Programme of the European Union.

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Reference List

(1) Hansen A, Lipsky PE, Dorner T. Immunopathogenesis of primary Sjogren’s syndrome:

implications for disease management and therapy. Curr Opin Rheumatol 2005; 17(5):558-65.

(2) Voulgarelis M, Dafni UG, Isenberg DA, Moutsopoulos HM. Malignant lymphoma in primary

Sjogren’s syndrome: a multicenter, retrospective, clinical study by the European Concerted Action

on Sjogren’s Syndrome. Arthritis Rheum 1999; 42(8):1765-72.

(3) Westermark P, Benson MD, Buxbaum JN, Cohen AS, Frangione B, Ikeda S et al. Amyloid fibril

protein nomenclature -2002. Amyloid 2002; 9(3):197-200.

(4) Merlini G, Bellotti V. Molecular mechanisms of amyloidosis. N Engl J Med 2003; 349(6):583-96.

(5) Falk RH, Comenzo RL, Skinner M. The systemic amyloidoses. N Engl J Med 1997; 337(13):898-

909.

(6) Ooms V, Decupere M, Lerut E, Vanrenterghem Y, Kuypers DR. Secondary renal amyloidosis due

to long-standing tubulointerstitial nephritis in a patient with Sjogren syndrome. Am J Kidney

Dis 2005; 46(5):e75-e80.

(7) Delevaux I, Andre M, Amoura Z, Kemeny JL, Piette JC, Aumaitre O. Concomitant diagnosis of

primary Sjogren’s syndrome and systemic AL amyloidosis. Ann Rheum Dis 2001; 60(7):694-5.

(8) Parambil JG, Myers JL, Lindell RM, Matteson EL, Ryu JH. Interstitial lung disease in primary

Sjogren syndrome. Chest 2006; 130(5):1489-95.

(9) Kambouchner M, Godmer P, Guillevin L, Raphael M, Droz D, Martin A. Low grade marginal zone

B cell lymphoma of the breast associated with localised amyloidosis and corpora amylacea in a

woman with long standing primary Sjogren’s syndrome. J Clin Pathol 2003; 56(1):74-7.

(10) Haraguchi H, Ohashi K, Yamada M, Hasegawa M, Maeda S, Komatsuzaki A. Primary localized

nodular tongue amyloidosis associated with Sjogren’s syndrome. ORL J Otorhinolaryngol Relat

Spec 1997; 59(1):60-3.

(11) Yoneyama K, Tochigi N, Oikawa A, Shinkai H, Utani A. Primary localized cutaneous nodular

amyloidosis in a patient with Sjogren’s syndrome: a review of the literature. J Dermatol 2005;

32(2):120-3.

(12) Srivastava M. Primary cutaneous nodular amyloidosis in a patient with Sjogren’s syndrome. J

Drugs Dermatol 2006; 5(3):279-80.

(13) Konishi A, Fukuoka M, Nishimura Y. Primary localized cutaneous amyloidosis with unusual

clinical features in a patient with Sjogren’s syndrome. J Dermatol 2007; 34(6):394-6.

(14) Fox RI. Sjogren’s syndrome. Lancet 2005; 366(9482):321-31.

(15) Mitsias DI, Kapsogeorgou EK, Moutsopoulos HM. Sjogren’s syndrome: why autoimmune

epithelitis? Oral Dis 2006; 12(6):523-32.

(16) Praprotnik S, Tomsic M, Perkovic T, Vizjak A. Is Sjogren’s syndrome involved in the formation of

localised nodular amyloidosis? Clin Exp Rheumatol 2001; 19(6):735-7.

(17) Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE et al.

Classification criteria for Sjogren’s syndrome: a revised version of the European criteria proposed

by the American-European Consensus Group. Ann Rheum Dis 2002; 61(6):554-8.

(18) Kebbel A, Rocken C. Immunohistochemical classification of amyloid in surgical pathology

revisited. Am J Surg Pathol 2006; 30(6):673-83.

(19) Novak L, Cook WJ, Herrera GA, Sanders PW. AL-amyloidosis is underdiagnosed in renal biopsies.

Nephrol Dial Transplant 2004; 19(12):3050-3.

(20) Gottenberg JE, Aucouturier F, Goetz J, Sordet C, Jahn I, Busson M et al. Serum immunoglobulin

free light chain assessment in rheumatoid arthritis and primary Sjogren’s syndrome. Ann Rheum

Dis 2007; 66(1):23-7.

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(21) Katzmann JA, Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR et al. Serum reference

intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains:

relative sensitivity for detection of monoclonal light chains. Clin Chem 2002; 48(9):1437-44.

Chapter 7

Summary and general discussion

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Summary

Sjögren’s syndrome (SS) is a systemic autoimmune disease characterised by chronic

inflammation of the salivary and lacrimal glands, resulting in complaints of xerostomia and

keratoconjunctivitis sicca in about 95% of the patients. These symptoms are frequently

accompanied by extraglandular manifestations, and 85% of the patients suffer from severe

fatigue.(1) Furthermore, the presence of SS has a large impact on health related quality of

life (HR-QoL), employment and disability.

Yet, no causal systemic treatment is available in SS and therefore only symptomatic

treatment can be given. Currently, biological agents have been introduced in various systemic

autoimmune diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus

(SLE). However, no biological agent has been approved thus far for the treatment of SS,

but several phase II and III studies have recently been completed or are currently being

conducted. The effect of treatment with biological agents is aimed at reducing disease

activity and to slow down progression of SS.

In the research described in this thesis the impact of SS on quality of life has been

evaluated, the different approved and experimental treatment options have been reviewed,

existing and new tools to evaluate treatment were assessed and treatment results with anti-

CD20 monoclonal antibodies (rituximab) are presented.

Chapter 2 describes HR-QoL, employment and disability in patients with primary (pSS)

and secondary (sSS) SS, compared to data available from the general Dutch population.

A questionnaire was sent to the total cohort of SS patients within the University Medical

Center Groningen that is seen for scheduled follow-up. 195 out of 235 patients (83%)

responded. The results revealed that SS has a large impact on HR-QOL, employment and

disability as reflected by lower Short Form-36 (SF-36) scores (measuring subjective well-

being), lower employment rates and higher disability rates in SS patients when compared to

the general Dutch population. In addition, physical functioning, bodily pain and general health

were worse in sSS than in pSS patients. The results of this trial underscore the necessity for

the development of causal treatment for SS.

Therefore, in chapter 3 an overview is given of the trials performed in SS with biological

agents up to 2006 and future perspectives are presented. The gain in knowledge regarding

the cellular mechanisms of T and B lymphocyte activity in the pathogenesis of SS and the

current availability of various biological agents (anti-TNF-α, IFN-α, anti-CD20, and anti-

CD22) have resulted in new possibilities for therapeutic intervention. In SS, various phase I

and II studies have been performed to evaluate these biologicals. Currently, B cell directed

therapies, and especially the use of anti-CD20 monoclonal antibodies, have been shown to

be more promising than T cell related therapies. In the near future a large role for treatment

with biologicals for SS is expected. Larger phase II and III trials are necessary to confirm

these first promising results.

In general, evaluation of a new treatment modality requires well defined and usable tools

to evaluate the effect of treatment. Chapter 4a gives a general overview of existing tools

for evaluation of treatment for diseases affecting salivary glands. Assessments of salivary

gland function (sialometry, sialochemistry) and histopathological examination of salivary

gland biopsies provide powerful tools to diagnose diseases affecting the salivary glands, to

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assess disease progression and to evaluate treatment. More general tools are subjective

questionnaires (e.g., visual analogue scale (VAS) scores, Multidimensional Fatigue Inventory

(MFI) score and SF-36) and serological parameters.

Chapter 4b describes the development of a new evaluation tool, the genomic and

proteonomic profile of whole saliva. In the study described in this chapter, the profiles for

SS patients were compared to healthy age and sex matched controls. This preliminary study

indicated that both glandular and whole saliva from pSS patients contain molecular signatures

that reflect damaged glandular cells and an activated immune response. Whole saliva was

shown to be more useful in SS diagnostics than parotid and submandibular/sublingual saliva.

The candidate proteonomic and genomic biomarkers found in whole saliva may improve the

clinical detection of pSS once they have been further validated in a larger group of patients.

The evaluation tools described in chapter 4 were used in evaluating treatment with

rituximab, described in chapter 5. In chapter 5a a study is described assessing the efficacy

and safety of (re)treatment of SS patients with rituximab after extended follow-up (mean

follow-up 57 weeks) of B cell depletion therapy. Included were 8 early pSS patients and

7 pSS patients with a mucosa-associated lymphoid tissue (MALT)-type lymphoma (MALT/

pSS). Rituximab was effective for 6-9 months in pSS patients and, probably, even longer

in MALT/pSS patients. Retreatment of 5 pSS patients resulted in a comparable beneficial

effect as observed after the first course. Development of serum sickness-like disorder in

27% of pSS patients indicated that higher doses of corticosteroids might be needed during

rituximab treatment.

In chapter 5b the results of histopathological evaluation of parotid tissue after rituximab

treatment were correlated with clinical results of parotid function in order to evaluate

rituximab treatment on a more fundamental level. Sequential parotid biopsies before and 12

weeks after rituximab treatment in pSS patients demonstrated histopathological evidence

of reduced glandular inflammation and redifferentiation of lymphoepithelial duct lesions to

regular striated ducts as a putative morphological correlate of increased parotid flow and

normalization of salivary sodium content. These histopathological findings underline the

efficacy of B cell depletion and prove the potential for glandular restoration in SS. This

study was performed as a pilot in the 5 pSS patients that received retreatment described

in chapter 5b. Analysis of larger groups of patients biopsied before and after rituximab

treatment are necessary to confirm these first results.

Based on these promising results, a randomized double-blind placebo-controlled trial

was performed (chapter 5c). In this trial 30 pSS patients were included, of which 20 were

treated with rituximab, while 10 patients received placebo. All 30 patients received an

additional dose of corticosteroids in order to prevent the development of side effects. In

this trial, B cell depletion led to improvement of objective and subjective parameters of

disease activity. Salivary function improved, fatigue diminished, extraglandular manifestations

improved. Most improvements were seen 12 to 36 weeks after treatment. These promising

results suggest that a larger phase III trial should be performed in order to receive approval

for rituximab treatment of SS.

Although SS is considered to be a T lymphocyte mediated disease, there are more and more

signs that the role of the B cells should not be underestimated.(2) The description of the

cases described in chapter 6 has deepened our insight into the B cell component of SS.

In this chapter, we retrospectively evaluated 8 patients with the combination of SS and

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localized cutaneous amyloidosis. The databases of 3 amyloidosis centres (Italy; University of

Pavia, Germany; University of Heidelberg and the Netherlands; Medical Center Groningen)

were searched in order to find this rare combination. It was likely that AL amyloid was the

actual type in all 8 patients, which is an immunoglobulin light chain associated amyloid, locally

produced by a light chain-restricted plasma cell population in the skin. The combination of

cutaneous amyloid and SS appeared to be a distinct disease entity reflecting a particular and

benign part of the polymorphic spectre of lymphoproliferative diseases related to SS.

General discussion

Sjögren syndrome: is there a need for treatment and which treatment is available?

SS is known to affect patients’ physical, psychological and social functioning (3), but the

impact of SS on health-related quality of life (HR-QOL), and especially on employment

and disability, has not been studied extensively before. However, this information is

necessary to interpret the burden of the disease and also to gain insight into the necessity

for treatment. Therefore, the analysis described in chapter 2 was performed. Comparable

to other autoimmune diseases, SS has a large impact on HR-QOL, employment and

disability as reflected by lower SF-36 scores and employment rates, and higher disability

rates when compared with the general Dutch population. The impact on socioeconomic

status described in chapter 2 justifies further research on biologicals in the treatment of SS,

even though these treatments are expensive and intensive. In addition, the overview of the

reports on biological treatment for SS (chapter 3) revealed that anti-CD20 (rituximab) is

the most promising biological agent so far.(4-6) The results of some of therapies targeting

TNF-α (infliximab, etanercept and adalimumab) and IFN-α were also promising in phase I

and II studies, but in larger placebo controlled randomized trials results were disappointing.

So, although the first results with rituximab seem promising, also regarding this biological

larger placebo controlled trials are needed to confirm these promising results (see section

on rituximab treatment). Moreover, as rituximab is a chimeric anti-CD20 agent that has

the inherent hazard of inducing serum sickness, humanized anti-CD20 (ocrelizumab) that

more recently has become available might, in potential, be an even more promising B cell

therapy. Another promising B cell directed therapy is anti-CD22 (epratuzumab). This agent

seemed to be effective in a small open-label trial, although to a lesser extent than rituximab

as it only partially depletes B cells.(7). Other potential targets for biological therapy include

cytokines such as IL-6 and BlyS (BAFF), interferons, adhesion molecules and chemokines.

No trials in SS have yet been performed with these biological therapies, however.

Which evaluation tools are useful?

With the increasing number of trials performed aiming to treat SS, there is a growing need

for more specific assessment parameters to monitor treatment effects, both subjectively

and objectively. For studies on intervention in SS, especially evaluation of the parotid gland

might be of use. Assessment of parotid secretory function (sialometry), composition of

parotid saliva (sialochemistry) and histological examination of parotid gland tissue (repeated

incisional biopsies) are routinely used in our setting to evaluate the effect of an intervention

therapy as a function of time. Also scintigraphy, functional MRI, PET scans and ultrasound

can be used repeatedly in evaluating the parotid gland. The diagnostic accuracy of the

latter tools is lower and these are therefore less often used in our setting for treatment

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evaluation. More general tools, but very valuable in evaluating intervention in SS, are

subjective questionnaires (e.g. VAS scores, MFI scores and SF-36) and serological parameters

such as rheumatoid factor and immunoglobulin levels, and B cell counts in the case of B cell

depletion therapy.

Furthermore, both glandular and whole saliva are easy to obtain and the first results

from studies on genomics and proteonomics (chapter 4b) showed valuable results. As a

continuation of this study, a validation paper reported on the discovery of highly specific

autoantibody biomarkers for pSS using protein microarray technology.(8) If the genomics

and proteonomics can be used in the future as diagnostic tools for SS and as tools for

monitoring the effect of treatment, for example rituximab treatment, in depth saliva

analysis might even replace more invasive diagnostic tools such as parotid biopsies, PET and

scintigraphy.

What about rituximab treatment?

Based on the promising results described in the review (chapter 3) and in the open label

phase II study (chapter 5a and 5b), a randomised, placebo-controlled trial with rituximab

was performed (chapter 5c). The results of the latter trial confirmed the promising results

of the phase II trials, but, also some criticism was raised related to the treatment of early

pSS patients without extraglandular manifestations with this biological. Because the long

term (side-)effects of treatment with biological agents in SS are not known yet, some SS

experts suggest to use treatment only for those SS patients with severe extraglandular

manifestations(9;10). However, we observed that patients with remaining glandular function

at the time of diagnosis benefit more from rituximab treatment than patients without any

function left. Thus, in our opinion patients with active disease, as reflected by high levels of

IgG and rheumatoid factor, increasing complaints of fatigue, and/or sicca complaints and/or

swelling of the parotid gland (but still having glandular function), are the preferred patients

to be treated with rituximab. Besides this group of early patients, also patients with severe

extraglandular manifestations may benefit significantly from treatment. Of course, the long-

term side effects of rituximab treatment have to be thoroughly investigated in larger phase

III trials before implementation of this biological as therapy for SS.

In contrast to patients with lymphoma or RA treated with rituximab, serum sickness

or serum sickness-like adverse events are more frequently reported in SS patients, with

a rate between 6% and 27%. (chapter 3) This initially unexpected finding may be due to

the use of different co-medication. Patients with RA and systemic lupus erythematosus

(SLE) usually receive higher doses of steroids or concomitantly immunosuppressive drugs

as compared with SS patients, which may prevent certain adverse events. In addition,

RA and SLE patients often have been treated with a wide range of medication (including

biological agents) before receiving treatment with rituximab, whereas SS patients are far

more medication-naïve at the time of rituximab treatment. We also observed in the trial

described in chapter 5c, as well as in our pilot trial, that patients who developed serum

sickness were more likely to have an active, early and progressive form of the disease.(6)

It is possible that such patients are more prone to develop serum sickness; however, such

patients might also be the ones that most likely benefit from rituximab therapy. Another

possibility is that SS patients may be more prone to develop and deposit immune complexes

because of hypergammaglobulinaemia and/or cryoglobulinemia.(4) Consequently, because

of the inherent risk of developing serum sickness (like) disease, we decided to increase

the steroid dose in the trial described in chapter 5c. Of the 30 included patients, only

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Table 1 Number of patients who actually received placebo or rituximab and the estimation of the patients and

the physicians.

Patient Patient Physician 1 Physician 1 Physician 2 Physician 2

True False True False True False

Rituximab (20) 16 4 18 2 17 3

Placebo (9) 7 2 8 1 8 1

Total (29) 23 (79%) 6 (21%) 26 (90%) 3 (10%) 25 (86%) 4 (14%)

one patient developed serum sickness-like disease (5%), which is considerably lower than

the incidence reported in our open-label study (27%).(6) Furthermore, HACA (human

antichimeric antibodies) development, which occurred in 27% of patients in our open-label

trial, was not found in the only patient who developed serum sickness-like disease. Based on

these findings, we would recommend administering 100 mg methylprednisolone immediately

prior to each infusion of rituximab. The oral regimen of prednisolone in the days following

each infusion differ between different trials and should be explored in future trials. The

administration of higher doses of prednisolone in the days following infusion, such as is

performed during lymphoma treatment, should also be considered, because most lymphoma

patients are, as SS patients, medication-naïve at time of rituximab treatment, and no serum

sickness has been reported in these patients.

Retreatment with rituximab resulted in a positive effect comparable to that of the first

treatment with this biological (chapter 5a). Therefore, offering patients maintenance therapy

with rituximab infusions every 6 to 9 months may be a reasonable approach. Advantages of

maintenance therapy might be a reduction or even arrest of disease progression and better

quality of life for a long period. A threat might be the, so far unknown, long term side

effects of repeated B cell depletion. The timing of retreatment could be based on return of

symptoms, however, retreatment just before return of symptoms would even be better. A

prediction model based on the results of our placebo controlled trial, showed that levels of

rheumatoid factor could be a good predictor for return of subjective symptoms such as dry

mouth and fatigue (unpublished results). However, these preliminary results were based on

20 pSS patients and, therefore, in future trials, attention should be paid to the correlation

between objective and subjective symptoms. We even like to pose that such a correlation

might provide a base for selecting the most optimal retreatment schedule. Probably, for

each patient an individual time scheme has to be made because we observed that the time

period in which rituximab reduced SS related symptoms/complaints differed considerably

between patients.

The dose of rituximab that patients should receive during maintenance treatment should

also be investigated. Based on the positive results after 2 infusions of 375mg/m² (which

is in total about 1000 mg) as reported by Devauchelle et al.(5), probably even only one

infusion of 1000 mg could be sufficient. Another issue concerns the question which group

of patients should be offered retreatment. In RA patients, results of trials on retreatment

of non-responders to first treatment are not conclusive. Thurlings et al. (11) reported that

only responders to the first treatment benefit from retreatment, while Vital et al. reports

that retreatment of non-responders before circulating plasma cells return to baseline

levels enhances B cell depletion and results in a better clinical response.(12) With respect

to SS, criteria for defining responders versus non-responders should first be formulated

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and validated and results of retreatment of both responders and non-responders should be

evaluated in future trials.

As a general rule, a placebo effect should not be underestimated in clinical trials with

a long follow up period. In order to obtain some insight into a placebo effect in a clinical

trial with only 30 patients (chapter 5c) all patients were asked after 24 weeks by mail if

they thought they received placebo or rituximab and the reason why they thought to

have received the active drug or placebo. One patient did not respond and was therefore

excluded from this analysis. Both study coordinators (physicians of the departments of

rheumatology and oral and maxillofacial surgery), who regularly assessed the patients and

who were blinded for the study medication, also guessed whether the patient had used

rituximab or placebo. In 23 out of 29 patients estimation of treatment was correct for both

physicians. The physicians correctly scored treatment modality of 25 and 26 patients out of

the 29 patients, respectively (Tables 1 and 2).

In conclusion, both the blinded patients and doctors could quite accurately estimate if a

patient received placebo or rituximab. Therefore, the placebo effect in this particular study

is small which gives us an additional hint that rituximab is an effective treatment for SS.

Role of B cells

The classical view on the role of B cells in immunity is focused on the production of

antibodies and autoantibodies in the case of autoimmune diseases. However, over the past

years the role of B cells seems to have acquired much more dimensions such as regulating

T cell subsets and dendritic cells through cytokine production, activation of T cells and

antigen presentation to T cells.(13;14) As other autoimmune diseases, SS is long considered

to be a T-lymphocyte mediated disease, however, in the light of these new developments

the role of B cells might be more prominent than thought in the past. The promising results

of B cell depletion therapy in SS also support the theory that there is a role for B cells in

the pathogenesis of SS. E.g., cutaneous nodular amyloidosis in SS seems to be the result

of a benign clonal proliferation of plasma cells in the skin that is part of the spectre of

lymphoproliferative diseases associated with SS. Despite its rare occurrence, 16 cases of

cutaneous amyloidosis have been reported in patients with SS, which is about 25% of the

reported cases of cutaneous amyloidosis These cases and the description of the cases

described in chapter 6 support the role of the B cell in SS.

Future perspectives

Today, SS is diagnosed more and more in an early stage of the disease. Screening might

become much easier if, in the future, e.g., the proteonomic profile can be used for diagnosis.

Only one drop of saliva might be sufficient for diagnostics and/or treatment evaluation.

Today no causal treatment is available, however, so far, the performed trials revealed that

B cell depletion with rituximab is probably the most effective therapy available to date. Also

our randomized double-blind placebo-controlled trial (chapter 5c) with rituximab treatment

showed promising results. A trial investigating retreatment of all patients involved in that

trial is in progress. Focus of that study will be a longer follow up period (64 weeks), the

effect of retreatment and the effect of treatment in patients who have received initially

a placebo. A histopathological study of parotid gland biopsies before and after rituximab

treatment of the patients described in chapter 5c has also been initiated and hopefully

confirms our clinical findings and the results of our pilot study on histopathological effects

of rituximab treatment (chapter 5b).

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Besides the already performed phase II trials, larger phase III trials are needed before

approval can be obtained for rituximab treatment in SS patients. In these larger phase III

studies, additional attention should be paid to the long term side effects, possibility of

retreatment, and the oral dose of prednisolone during the days after each infusion. We

also like to pose that rituximab treatment is especially effective for patients with active

disease, extraglandular manifestations and/or remaining salivary gland secretory potential.

To confirm these hypotheses, in future larger trials less strict inclusion criteria related

to baseline salivary gland function and a larger number of patients are needed. In order

to define treatment protocols, criteria regarding responders/non-responders have to be

implemented. Studies regarding disease activity scores are currently being performed and

are also important for future treatment protocols.

In addition to phase III rituximab trials, also other types of B cell depletion therapies

should be investigated including completely humanized anti-CD20, anti-CD22 and anti-

BAFF. To our opinion, there is a large role in the future for biologicals in the treatment of SS

which could add substantially to a good quality if life of SS patients.

Table 2 Number of correct estimations. Maximum score is 3: patient and both physicians scored correct.

Number of correct estimations 0 1 2 3

Number of patients 1 (3%) 2 (7%) 4 (14%) 22 (76%)

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Reference List

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(2) Looney RJ. Will targeting B cells be the answer for Sjogren’s syndrome? Arthritis Rheum 2007;

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(3) Bjerrum K, Prause JU. Primary Sjogren’s syndrome: a subjective description of the disease. Clin Exp

Rheumatol 1990; 8(3):283-8.

(4) Dass S, Bowman SJ, Vital EM, Ikeda K, Pease CT, Hamburger J et al. Reduction of fatigue in Sjogren’s

syndrome with rituximab: results of a randomised, double-blind, placebo controlled pilot study.

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(5) Devauchelle-Pensec V, Pennec Y, Morvan J, Pers JO, Daridon C, Jousse-Joulin S et al. Improvement

of Sjogren’s syndrome after two infusions of rituximab (anti-CD20). Arthritis Rheum 2007;

57(2):310-7.

(6) Pijpe J, van Imhoff GW, Spijkervet FKL, Roodenburg JLN, Wolbink GJ, Mansour K et al. Rituximab

treatment in patients with primary Sjögren’s syndrome: An open-label phase II study. Arthritis

Rheum 2005; 52(9):2740-50.

(7) Steinfeld SD, Tant L, Burmester GR, Teoh NK, Wegener WA, Goldenberg DM et al. Epratuzumab

(humanized anti-CD22 antibody) in primary Sjogren‘s syndrome: An open-label Phase I/II study.

Arthritis Res Ther 2006; 8(4):R129.

(8) Hu S, Vissink A, Arellano M, Kallenberg CG, Wong DT. Salivary autoantibody biomarkers for

Sjögren‘s syndrome. Mol Cell Proteomics. Submitted 2009.

(9) Isaksen K, Jonsson R, Omdal R. Anti-CD20 treatment in primary Sjogren‘s syndrome. Scand J

Immunol 2008; 68(6):554-64.

(10) Seror R, Sordet C, Guillevin L, Hachulla E, Masson C, Ittah M et al. Tolerance and efficacy of

rituximab and changes in serum B cell biomarkers in patients with systemic complications of

primary Sjogren‘s syndrome. Ann Rheum Dis 2007; 66(3):351-7.

(11) Thurlings RM, Vos K, Gerlag DM, Tak PP. Disease activity-guided rituximab therapy in rheumatoid

arthritis: the effects of re-treatment in initial nonresponders versus initial responders. Arthritis

Rheum 2008; 58(12):3657-64.

(12) Vital EM, Dass S, Buch MH, Horner E.A., Goeb V., Rawstron A.C. et al. How to manage non-response

to rituximab: predictors and outcome of retreatment provide data for a treatment algorithm. Ann

Rheum Dis 68[suppl3], 77. 2009.

(13) Anolik JH, Looney RJ, Lund FE, Randall TD, Sanz I. Insights into the heterogeneity of human B

cells: diverse functions, roles in autoimmunity, and use as therapeutic targets. Immunol Res 2009.

(14) Porakishvili N, Mageed R, Jamin C, Pers JO, Kulikova N, Renaudineau Y et al. Recent progress in the

understanding of B-cell functions in autoimmunity. Scand J Immunol 2001; 54(1-2):30-8.

Chapter 8

Samenvatting

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Samenvatting

Het syndroom van Sjögren (SS) is een auto-immuunziekte die wordt gekarakteriseerd door

een chronische ontsteking van onder andere de speeksel- en traanklieren. Deze ontsteking

leidt bij 95% van de patiënten tot klachten van een droge mond (xerostomie) en droge

ogen (keratoconjunctivitis sicca). Dit beeld wordt het primair syndroom van Sjögren (pSS)

genoemd. Wanneer de aandoening gepaard gaat met een andere auto-immuunziekte zoals

reumatoïde artritis (RA) of systemische lupus erythematosus (SLE) spreken we van een

secundair syndroom van Sjögren (sSS). De mond- en oogproblemen worden vaak vergezeld

door klachten buiten de klieren (extraglandulair) zoals nier- en long problemen, ontsteking van

gewrichten (artritis), pijnlijke gewrichten (artralgie) en ontsteking van bloedvaten (vasculitis).

Daarnaast ondervindt 85% van de patiënten klachten van ernstige vermoeidheid. Tenslotte

heeft SS een grote impact op de ziekte gerelateerde kwaliteit van leven.

De huidige behandelingen voor SS onderdrukken alleen, in wisselende mate, de symptomen

van de ziekte. Sinds een aantal jaren wordt therapie met biologicals toegepast voor auto-

immuunziekten, zoals RA. Bij therapie met biologicals wordt gepoogd op celniveau in te

grijpen in de ontstaanswijze (pathofysiologie) van de ziekte. Voor SS zijn echter nog geen

goedgekeurde therapieën met biologicals beschikbaar voor klinische toepassing. Inmiddels zijn

wel een aantal fase I en II studies uitgevoerd of in uitvoering waarin het nut van een dergelijke

therapie voor SS wordt onderzocht.

In het onderzoek beschreven in dit proefschrift wordt de invloed van SS op de kwaliteit

van leven geëvalueerd, worden de tot op heden toegepaste goedgekeurde en experimentele

behandelingen voor SS beschreven, worden bestaande en nieuwe instrumenten voor het

evalueren van de behandeling van SS besproken en worden de effecten van behandeling met

rituximab, een antilichaam gericht tegen bepaalde witte bloedcellen (B cellen), in SS patiënten

geëvalueerd.

In hoofdstuk 2 wordt de aan de ziekte gerelateerde kwaliteit van leven en de invloed op werk

en arbeids(on)geschiktheid van pSS en sSS patiënten beschreven. De gegevens van SS patiënten

werden vergeleken met data afkomstig uit de gemiddelde Nederlandse populatie. Aan het

gehele cohort van SS patiënten, die regelmatig voor controle in het Universitair Medisch

Centrum Groningen werd gezien, werd een vragenlijst toegestuurd. 195 van de 235 patiënten

(83%) bleken bereid te zijn aan dit onderzoek deel te nemen en stuurden de ingevulde

vragenlijst terug. Analyse van de resultaten toonde aan dat SS een grote invloed had op de aan

de ziekte gerelateerde kwaliteit van leven en op werk en arbeids(on)geschiktheid. In vergelijking

met de gemiddelde scores van de Nederlandse populatie scoorden SS patiënten lager op

het Short Form-36 (SF-36; deze vragenlijst scoort het subjectieve welbevinden), bleek het

percentage SS patiënten dat werkte lager te zijn en was het percentage arbeidsongeschiktheid

onder SS patiënten beduidend hoger dan in de Nederlandse populatie. Patiënten met sSS

scoorden slechter dan patiënten met pSS op de gebieden fysiek functioneren, lichamelijke pijn

en algemene gezondheid van de SF-36 vragenlijst. De resultaten van dit onderzoek benadrukken

de noodzaak tot het ontwikkelen van een meer causale behandeling voor SS.

In hoofdstuk 3 wordt een overzicht gepresenteerd van de tot en met 2006 gepubliceerde

resultaten van onderzoek verricht naar de effecten van therapie met biologicals in de

behandeling van SS. Tevens worden in dit hoofdstuk de toekomstperspectieven betreffende

de behandeling van SS met deze therapieën geschetst. Toegenomen inzicht in de werking op

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celniveau van witte bloedcellen (cellulaire mechanismen van T en B cel activiteit), toegenomen

kennis van de pathofysiologie van SS en het beschikbaar zijn van een aantal therapieën met

biologicals (anti-TNF-α, anti-CD20, anti-CD22) hebben geresulteerd in nieuwe mogelijkheden

voor therapeutische interventie. Inmiddels zijn een aantal fase I en II onderzoeken uitgevoerd

om de effectiviteit en veiligheid van therapieën met biologicals voor SS te evalueren.

Momenteel lijken B cel gerichte therapieën, in het bijzonder de therapie waarbij anti-CD20

antilichamen worden toegepast, veelbelovend te zijn. Aangezien de uitkomsten van de tot

op heden gerapporteerde inventariserende studies met B cel gerichte therapieën hoopvol

zijn, is het de inschatting dat in de nabije toekomst therapieën met biologicals een belangrijke

rol zullen gaan spelen bij de behandeling van SS. Alvorens deze therapieën algemeen kunnen

worden toegepast, moeten eerst grotere fase II en III studies worden verricht om de

gerapporteerde resultaten te bevestigen.

Evaluatie van een nieuwe therapie vereist goed gedefinieerde en gebruiksvriendelijke

instrumenten om het effect van de behandeling te beoordelen. In hoofdstuk 4a wordt een

overzicht gegeven van de bestaande instrumenten waarmee het effect van een therapie

gericht op speekselklier gerelateerde ziekten kan worden geëvalueerd. Evaluatie van de

speekselklierfunctie (hoeveelheid speeksel (sialometrie) en samenstelling (sialochemie)) en

onderzoek van de biopten op weefselniveau (histopathologie) van de speekselklieren lijken

zeer geschikte instrumenten om zowel speekselklier gerelateerde ziekten te diagnosticeren

als de progressie en behandeling van de onderliggende aandoening te evalueren. Daarnaast

is het zinvol om meer algemene instrumenten, zoals subjectieve vragenlijsten (bijvoorbeeld

visual analogue scale (VAS) scores, Multidimensional Fatigue Inventory (MFI) score en SF-36)

en serologische parameters, zoals gehaltes van autoantilichamen in het bloed, bij de evaluatie

van het effect van een bepaalde therapie op SS te betrekken.

In hoofdstuk 4b wordt de ontwikkeling van een nieuw evaluatie instrument beschreven:

analyse van het genetische en eiwitprofiel van totaal speeksel. In het in dit hoofdstuk

beschreven onderzoek werden de genoemde speeksel profielen van SS patiënten vergeleken

met die van gezonde mensen van dezelfde leeftijd en hetzelfde geslacht. Uit het onderzoek

kwam naar voren dat zowel het speeksel van specifieke klieren als totaal speeksel van pSS

patiënten moleculaire profielen bevat die weergeven dat de speekselklier beschadigd is en

het immuunsysteem geactiveerd is. De eiwit en genetische biomarkers die werden gevonden

in het totaal speeksel kunnen mogelijk van belang zijn voor de vroegdiagnostiek van pSS.

Hiervoor moeten de gevonden markers worden gevalideerd in grotere groepen patiënten

en worden afgezet tegen het profiel van patiënten met andere auto-immuunziekten, zoals

reumatoïde artritis, SLE en sSS.

De in hoofdstuk 4 beschreven evaluatie instrumenten zijn gebruikt om de behandeling met

rituximab (hoofdstuk 5) te evalueren.

In hoofdstuk 5a wordt een studie beschreven waarin de effectiviteit en de veiligheid

van (her)behandeling met rituximab van patiënten met SS wordt geëvalueerd. De follow

up bedroeg gemiddeld 57 weken. Acht patiënten met vroege pSS en 7 patiënten met een

mucosa-associated lymphoid tissue (MALT)-type lymfoom en pSS (MALT/pSS) werden

geïncludeerd. De behandeling met rituximab bleek ongeveer 6-9 maanden effectief voor

vroege pSS patiënten en langer voor de MALT/pSS patiënten. Herbehandeling van de 5 pSS

patiënten die geen serumziekte hadden ontwikkeld, resulteerde in een vergelijkbaar positief

effect zoals werd gezien na de eerste behandeling. Het ontwikkelen van een serumziekte-

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achtig beeld, veroorzaakt door de ontwikkeling van antilichamen tegen rituximab, bij 3 van de

8 behandelde patiënten zou kunnen beteken dat er een hogere dosis corticosteroïden nodig

is tijdens de behandeling met rituximab.

In hoofdstuk 5b wordt een studie beschreven waarin de resultaten van de histopatho-

logische evaluatie van weefsel van de oorspeekselklier (parotis) na rituximab behandeling

werden gecorreleerd aan de parotisfunctie met als doel om een structuur-functie analyse van

de behandeling met rituximab te kunnen verrichten. Sequentiële parotisbiopten, vóór en 12

weken ná behandeling met rituximab van vroege pSS patiënten, toonden een afname van de

ontsteking en herstel van de in het ontstekingsproces veranderde klierbuisjes. De toegenomen

speekselvloed van de parotisklier en de normalisatie van de natrium concentratie in het

parotisspeeksel zijn in overeenstemming met de op histopathologisch niveau waargenomen

veranderingen. Deze bevindingen onderstrepen de effectiviteit van B cel depleterende

therapie en duiden er op dat regeneratie van speekselklierweefsel mogelijk is bij SS. Het in dit

hoofdstuk beschreven onderzoek werd verricht bij de 5 patiënten die werden herbehandeld

met rituximab (zie hoofdstuk 5a). Analyse van grotere groepen patiënten waarbij een biopt

is genomen voor en na behandeling met rituximab is nodig om deze eerste resultaten te

bevestigen.

Gebaseerd op deze veelbelovende resultaten werd een dubbel-blinde placebo-gecontro-

leerde studie verricht (hoofdstuk 5c). In dit onderzoek werden 30 patiënten met een

vroege vorm van pSS geïncludeerd, waarvan 20 patiënten werden behandeld met rituximab

en 10 patiënten met een placebo. Alle 30 pSS patiënten kregen een hogere dosering

corticosteroïden dan de pSS patiënten in de eerdere inventariserende studie (hoofdstuk

5a) om het ontwikkelen van bijwerkingen, in het bijzonder een op serumziekte gelijkend

klachtenpatroon, te voorkomen. In dit onderzoek leidde B cel depletie tot verbetering van

zowel de objectieve als subjectieve parameters van de aan pSS gerelateerde ziekteactiviteit.

De speekselklierfunctie verbeterde, de vermoeidheid verminderde en de extraglandulaire

manifestaties namen af. De meeste verbeteringen werden 12 tot 36 weken na de start van de

behandeling met rituximab gezien. Deze veelbelovende resultaten suggereren dat het zinvol is

om een grotere fase III studie uit te voeren met als doel het verkrijgen van goedkeuring voor

behandeling met rituximab bij SS.

Hoewel SS wordt beschouwd als een ziekte waarbij met name T cellen betrokken zijn bij

het ontstaan van de afwijkingen, bestaan er steeds meer aanwijzingen dat de rol van de B

cellen niet moet worden onderschat. De beschrijving van de casus in hoofdstuk 6 vergroot

het inzicht in de betrokkenheid van een B cel component bij SS. In dit hoofdstuk wordt een

retrospectief onderzoek beschreven naar 8 patiënten met de combinatie van SS en een

lokale huidaandoening waarbij er neerslag plaatsvindt van eiwitten (gelokaliseerde cutane

amyloidose). In databases van 3 amyloidose centra (Italië: Universiteit van Pavia; Duitsland:

Universiteit van Heidelberg; Nederland: Universitair Medisch Centrum Groningen) werd

gezocht naar deze zeldzame combinatie. Meest waarschijnlijk was er sprake van AL type

amyloidose bij alle 8 SS patiënten. Dit is een lichte keten immunoglobuline geassocieerde

amyloidose waarbij deze ketens lokaal worden geproduceerd door plasma cellen in de huid

die uitsluitend lichte ketens produceren. Cutane AL amyloidose lijkt samen te hangen met

SS. Hiermee wordt een nieuw element toegevoegd aan het spectrum van lymfoproliferatieve

ziekten dat gerelateerd is aan SS.

In hoofdstuk 7 worden de algemene conclusies uit de verschillende onderzoeken gecombineerd,

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besproken en in een breder kader geplaatst. Tevens worden toekomstperspectieven geschetst

ten aanzien van de causale behandelmogelijkheden van SS.

Tegenwoordig wordt SS steeds vaker in een vroeg stadium van het ziekteproces gediag-

nosticeerd. Screening op SS zou in de toekomst kunnen worden vereenvoudigd wanneer

hiervoor bijvoorbeeld het eiwitprofiel van speeksel kan worden gebruikt.

Tot op heden is geen causale behandeling beschikbaar voor SS. Wel is aangetoond

dat het hebben van SS een grote invloed heeft op de kwaliteit van leven, op werk en

arbeidsgeschiktheid. Daarom is het van belang dat er onderzoek wordt gedaan naar nieuwe

behandelingen voor SS, ook al zijn deze behandelingen duur en intensief.

Uit literatuuronderzoek is gebleken dat behandelingen die gericht zijn op depletie van B

cellen het meest succesvol zijn. Ook de resultaten van een placebo gecontroleerde studie

(hoofdstuk 5c) lieten positieve effecten zien van behandeling met rituximab, een B cel

depleterende behandeling. Een probleem van een behandeling met rituximab is dat bij SS

patiënten veel vaker een serumziekte-achtig beeld wordt gezien dan bij patiënten met andere

auto-immuun aandoeningen, bijvoorbeeld RA en SLE. Een aantal hypotheses die dit verschil

zouden kunnen verklaren, worden uiteengezet in hoofdstuk 7. Toediening van prednisolon

lijkt de kans op het ontwikkelen van dit serumziekte-achtige beeld te verkleinen.

Herbehandeling van SS met rituximab lijkt even effectief te zijn als een eerste behandeling.

Momenteel loopt een studie waarbij alle 30 in hoofdstuk 5c beschreven pSS patiënten

worden herbehandeld met rituximab en waarbij een langere follow up periode (ruim 1

jaar) in acht wordt genomen. In deze studie krijgen alle patiënten, dus zowel de patiënten

die aanvankelijk rituximab kregen als ook de patiënten die een placebo hebben gekregen,

rituximab toegediend. Naast een beoordeling van het klachtenpatroon, en serologisch- en

speekselklierfunctieonderzoek, worden bij deze 30 pSS patiënten opnieuw parotisbiopten

genomen (vóór en/of 12 weken ná behandeling met rituximab). Deze biopten zullen

histopathologisch worden geanalyseerd. Met deze studie hopen we de in hoofdstuk 5b

beschreven resultaten te bevestigen.

Naast de al uitgevoerde fase II studies moeten grotere fase III studies worden verricht om

toestemming te krijgen voor routine behandeling van SS patiënten met rituximab. In deze

grote fase III studies zal aandacht moeten worden geschonken aan de lange termijn effecten

van rituximab, aan de mogelijkheid tot herbehandeling en aan het optimale doseringsschema

van prednisolon in de dagen van en na het toedienen van rituximab.

Op basis van de resultaten van het in dit proefschrift beschreven onderzoek kan gesteld

worden dat behandeling met rituximab effectief is bij pSS patiënten met een actief ziektebeeld

en/of met een restfunctie van de speekselklieren. Daarnaast is rituximab effectief voor de

behandeling van extraglandulaire manifestaties. Om deze stelling te bevestigen zullen bij

toekomstige grote(re) studies minder strikte inclusiecriteria moeten worden gehanteerd, dat

wil zeggen dat ook patiënten met een langere ziekteduur en/of een lagere speekselsecretie bij

aanvang van de studie moeten worden geïncludeerd. Voorts moeten, om behandelprotocollen

te kunnen opstellen, eerst algemeen geaccepteerde responder/non-responder criteria worden

opgesteld. Binnen dit kader worden momenteel studies uitgevoerd waarbij wordt gekeken

naar scores die ziekteactiviteit meten.

Naast de fase III studies met rituximab zouden ook andere op B cel gerichte therapieën

moeten worden onderzocht, zoals gehumaniseerd anti-CD20, anti-CD22 en anti-BAFF. In de

toekomst lijkt een grote rol weggelegd voor de therapie met biologicals in de behandeling van

SS. Dergelijke therapieën zouden substantieel kunnen bijdragen aan het verbeteren van de

kwaliteit van leven van de patiënten met SS.

Dankwoord

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Het is klaar!

Met hulp van veel mensen heb ik gewerkt aan het onderzoek beschreven in dit proefschrift.

Een aantal daarvan wil ik hier graag persoonlijk bedanken.

Allereerst wil ik de patiënten bedanken die hebben deelgenomen aan het onderzoek

beschreven in dit proefschrift.

Prof. dr. L.G.M. de Bont wil ik graag bedanken voor de mogelijkheid die ik heb gekregen om

dit onderzoek te combineren met de studie tandheelkunde op een heel prettige afdeling.

Prof. dr. A. Vissink, beste Arjan, jij bent als eerste promotor de afgelopen jaren van heel

dichtbij betrokken geweest bij het uitvoeren van dit onderzoek. Ik heb groot respect en

veel waardering voor de manier waarop je dit gedaan hebt. Je bent integer, snel, scherp,

laagdrempelig, je hebt overzicht. Deze eigenschappen maken dat ik mij geen betere eerste

promotor had kunnen wensen!

Prof. dr. C.G.M. Kallenberg, beste Cees, je kennis en kunde op het immunologische vlak

waren onmisbaar bij het opzetten en uitvoeren van de verschillende studies beschreven

in dit proefschrift. Je beschikt over een onuitputtelijk stroom ideeën en daardoor ben je

voor mij een zeer motiverende promotor geweest. Ik wil je ook bedanken voor de snelheid

waarmee je mijn manuscripten van (altijd zeer nuttig) commentaar voorzag.

Dr. H. Bootsma, beste Hendrika, samen hebben we heel wat uren gewerkt aan de opzet

en uitvoering van de klinische studies en ik heb veel waardering voor je praktische en

doortastende aanpak hierbij. We hebben samen veel congressen bezocht, deze waren

leerzaam maar bovenal ook altijd erg gezellig! Bedankt hiervoor.

Dr. F.K.L. Spijkervet, beste Fred, jouw inbreng lag ook met name op het klinische vlak, maar

dan het kaakchirurgische deel hiervan. Jij hebt alle parotis biopten uitgevoerd en je hebt je

ook gebogen over de logistiek van de verschillende studies. Tijdens de polimiddagen mocht

ik altijd een beroep op je doen voor overleg. Bedankt hiervoor.

De leden van de beoordelingscommisie, prof. dr. J.C. Kluin-Nelemans, prof. dr. I. van der

Waal en prof. dr. P.P. Tak, wil ik bedanken voor de voortvarende beoordeling van het

manuscript.

Dr. W.W.I. Kalk en dr. J. Pijpe, beste Wouter en Justin, het was mijn taak en uitdaging om

de door jullie zo goed opgezette onderzoekslijn voort te zetten. Ik heb dit met veel plezier

gedaan en geef nu het stokje door aan drs. P.M. Meiners en drs. R.P.E. Pollard. Petra, bedankt

voor je grote bijdrage aan de klinische studies beschreven in dit proefschrift, ik vind het

erg leuk dat jij nu de vervolgstudies opzet en uitvoert. Rodney, jij richt je met name op het

histologische deel van het onderzoek. Het is heel fijn dat ook dit deel van de onderzoekslijn

weer helemaal lopende is. Daarnaast was het erg gezellig om een kamer met je te delen, ook

al had je het afgelopen zomer soms best zwaar met twee zwangere kamergenoten…. Ik wil

jullie veel succes toewensen met het vervolgonderzoek in deze leuke onderzoeksgroep.

Janita Bulthuis-Kuiper, jij was onmisbaar bij alle logistiek van de rituximab studie. Jij hebt

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heel wat afspraken gepland, patiënten gebeld en vragenlijsten ingevoerd. Heel erg bedankt

hiervoor!

Ik wil alle medewerkers op de polikliniek kaakchirurgie, in het bijzonder Jenny van den

Akker, Piet Haanstra, Miranda Been en dr. Monique Stokman, bedanken voor de hulp bij de

vele patiëntenonderzoeken.

De afdeling reumatologie en klinische immunologie wil ik bedanken voor de prettige

samenwerking. In het bijzonder wil ik dr. Liesbeth Brouwer bedanken voor de medewerking

aan het klinische deel van de studies en dr. Bouke Hazenberg wil ik bedanken voor de leuke

samenwerking welke heeft geresulteerd in het artikel over amyloidose en Sjögren. Eefke

Eppinga, Diana Nijborg, Janny Havinga en Kiki Bugter wil ik bedanken voor alle logistieke

ondersteuning.

Marcel van der Leij, Siep Postma en Bessel Schaap, bedankt voor alle FACS analyses die jullie

hebben uitgevoerd. Dit was een hele klus. Drs. J. Bijzet, dr. W. Abdulahad, prof. dr. P.C.

Limburg, dr. C. Roozendaal en dr. J. Westra. Beste Johan, Wayel, Piet, Caroline en Hannie,

bedankt voor de goede samenwerking.

Drs. N. Kamminga, dr. K. Mansour, prof. dr. P.M. Kluin, dr. J.E. Van der Wal, dr. G.W. van

Imhoff, prof. dr. N. Bos, prof. dr. F. Kroese en drs. N. Hamza, beste Nicole, Khaled, Philip,

Jaqueline, Gustaaf, Nico, Frans en Nishat, bedankt voor alle boeiende discussies tijdens de

bijeenkomsten van de Sjögren werkgroep en bedankt voor de plezierige samenwerking en

jullie bijdrage aan de verschillende studies.

Prof. D. Wong and dr. S. Hu, dear David and Shen, I would like to thank you for the pleasant

cooperation in the proteonomics and genomics project. This resulted in a chapter in this

thesis.

De maatschap kaakchirurgie uit het Medisch Centrum Leeuwarden, bedankt voor het

keuzecoschap wat ik bij jullie heb mogen lopen. Deze enthousiasmerende maanden hebben

mijn keuze om aan dit opleidingtraject te willen beginnen gemakkelijk gemaakt.

Alle medeonderzoekers op de derde verdieping wil ik bedanken voor alle gezelligheid. Naast

de gezelligheid vond ik het ook prettig om alle tandheelkunde- en onderzoekservaringen

met jullie te kunnen delen.

Lisa Kempers, Karin Wolthuis, Nienke Jaeger en Harrie de Jonge, ook jullie bedankt voor

alle gezelligheid en natuurlijk ook voor de administratieve ondersteuning.

Drs. W. Nesse, beste Willem, samen zijn wij begonnen aan de studie tandheelkunde. Ik

ben heel blij dat ik dit met jou heb kunnen doen. We hebben op de faculteit heel veel leuke

momenten (en zelfs onze patiënten) kunnen delen. Het samen (pogen te) cementeren van

een kroon zal ik nooit vergeten… Bedankt dat jij mijn paranimf wil zijn.

Drs. S. Visscher-Langeveld, lieve Susan, wij hebben een paar jaar een kamer gedeeld op de

derde verdieping. Het is heel jammer dat alle ‘appelflap en deur dicht’ momenten nu voorbij

zijn, ik zal het missen, maar we vinden zeker een manier om deze momenten te vervangen!

Bedankt dat jij mijn paranimf wil zijn.

Dan

kwoo

rd

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Lieve Gerda en Wim, ik wil jullie bedanken voor de opvoeding die wij van jullie hebben

gekregen. Vanuit een warme en veilige thuisbasis hebben jullie ons altijd gestimuleerd en de

vrijheid gegeven om eigen keuzes te maken. Bedankt voor jullie onvoorwaardelijke liefde,

steun en interesse.

Lieve Annieka en Miriam, ik ben heel blij dat jullie mijn zusjes zijn! Bedankt voor jullie

belangstelling en alle gezellige momenten.

Lieve Albert, Marja, Menno, Mechteld, Judith, Erik, Yvo en alle verdere familie en vrienden,

bedankt voor alle gezellige (niet werk gerelateerde) momenten de afgelopen jaren, ik hoop

dat er nog vele zullen volgen.

Lieve Janwillem, wat is het leuk om samen met jou te zijn!

Lieve Nander en Borrit, ik geniet elke dag volop van jullie komst in ons leven!

Dankwoord155

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Curriculum vitae

Jiska Meijer werd 6 maart 1979 geboren te Vlaardingen. In 1997 deed ze eindexamen VWO aan

het Carolus Clusius College te Zwolle. Van september 1997 tot september 1998 studeerde zij

Industrieel Ontwerpen aan de Technische Universiteit Delft. In september 1998 startte zij met

de studie geneeskunde aan de Rijksuniversiteit Groningen. Haar artsenbul behaalde zij cum

laude in augustus 2004. Tijdens haar studie was Jiska actief in diverse commissies, zij vervulde

onder andere een tweejarige functie als studentlid van de faculteitsraad. In september 2004

startte zij als arts-onderzoeker op de afdeling Kaakchirurgie van het Universitair Medisch

Centrum Groningen. Van september 2005 tot en met april 2009 combineerde zij haar

promotieonderzoek met de studie tandheelkunde aan de Rijksuniversiteit Groningen. In april

2009 behaalde zij haar tandartsenbul cum laude. Sinds september 2009 is Jiska in opleiding

tot kaakchirurg.

Jiska woont samen met Janwillem Kocks en samen hebben zij twee zoons, Nander geboren op

20 februari 2008 en Borrit geboren op 14 september 2009.

J.M. Meijer

University Medical Center Groningen

Department of Oral and Maxillofacial Surgery

9700 RB Groningen

The Netherlands

j.m.meijer@kchir.umcg.nl